Method of efficiently establishing induced pluripotent stem cells

ABSTRACT

The present invention provides a method of improving the efficiency of establishment of induced pluripotent stem (iPS) cells, comprising inhibiting the p53 function in the step of somatic cell nuclear reprogramming. The inhibition of p53 function is achieved by bringing a substance selected from the group consisting of (1) chemical inhibitors of p53, (2) dominant negative mutants of p53 and nucleic acids that encode the same, (3) siRNAs and shRNAs against p53 and DNAs that encode the same, and (4) p53 pathway inhibitors, into contact with a somatic cell, and the like. The present invention also provides an agent for improving the efficiency of establishment of iPS cells, the agent comprising an inhibitor of p53 function, particularly (1) chemical inhibitors of p53, (2) dominant negative mutants of p53 and nucleic acids that encode the same, (3) siRNAs and shRNAs against p53 and DNAs that encode the same, and (4) p53 pathway inhibitors. The present invention further provides a method of producing an iPS cell, comprising bringing a nuclear reprogramming substance and an inhibitor of p53 function into contact with a somatic cell.

This application is based on U.S. provisional patent application Nos.61/076,487, 61/095,573, 61/194,700, 61/200,307 and 61/209,686, thecontents of which are hereby incorporated by reference.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY SUBMITTED

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: 44,409 bytes ASCII (Text) file named “706063SequenceListing.txt,” created Feb. 2, 2010.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a method of improving the efficiency ofestablishment of induced pluripotent stem (hereinafter referred to asiPS) cells and a drug therefor. More specifically, the present inventionrelates to a method of improving the efficiency of establishment of iPScells by inhibiting the p53 function in the step of somatic cell nuclearreprogramming, and an agent for improving the efficiency ofestablishment of iPS cells with an inhibitor of p53 function as anactive ingredient.

BACKGROUND OF THE INVENTION

In recent years, mouse and human iPS cells have been established oneafter another. Yamanaka et al. induced iPS cells by introducing theOct3/4, Sox2, Klf4 and c-Myc genes into fibroblasts derived from areporter mouse wherein the neomycin resistance gene is knocked-in intothe Fbx15 locus, and forcing the cells to express the genes (1,2). Okitaet al. (3) succeeded in establishing iPS cells (Nanog iPS cells) thatshow almost the same gene expression and epigenetic modification asthose in embryonic stem (ES) cells by producing a transgenic mousewherein the green fluorescent protein (GFP) and puromycin-resistancegenes are integrated into the locus of Nanog, whose expression is morelocalized in pluripotent cells than Fbx15 expression, forcing thefibroblasts derived from the mouse to express the above-mentioned 4genes, and selecting puromycin-resistant and GFP-positive cells. Similarresults were confirmed by other groups (4,5). Thereafter, it wasrevealed that iPS cells could also be produced with 3 factors other thanthe c-Myc gene (6). Furthermore, Yamanaka et al. succeeded inestablishing iPS cells by introducing the same 4 genes as those used inthe mouse into human skin fibroblasts (1,7). On the other hand, a groupof Thomson et al. produced human iPS cells using Nanog and Lin28 inplace of Klf4 and c-Myc (8,9). Park et al. (10) produced human iPS cellsusing TERT, which is known as the human cell immortalizing gene, and theSV40 large T antigen, in addition to the 4 factors Oct3/4, Sox2, Klf4and c-Myc. Hence, it has been demonstrated that iPS cells comparable toES cells in pluripotency can be produced in both humans and mice byintroducing defined factors into somatic cells.

However, the efficiency of iPS cell establishment is low at less than1%. Especially, a problem of extremely low efficiency of iPS cellestablishment occurs when they are produced by introducing 3 factors(Oct3/4, Sox2 and Klf4) other than c-Myc, which is feared to causetumorigenesis in tissues or individuals differentiated from iPS cells,into somatic cells.

REFERENCES CITED

-   1. WO 2007/069666 A1-   2. Takahashi, K. and Yamanaka, S., Cell, 126: 663-676 (2006)-   3. Okita, K. et al., Nature, 448: 313-317 (2007)-   4. Wernig, M. et al., Nature, 448: 318-324 (2007)-   5. Maherali, N. et al., Cell Stem Cell, 1: 55-70 (2007)-   6. Nakagawa, M. et al., Nat. Biotethnol., 26: 101-106 (2008)-   7. Takahashi, K. et al., Cell, 131: 861-872 (2007)-   8. WO 2008/118820 A2-   9. Yu, J. et al., Science, 318: 1917-1920 (2007)-   10. Park, I. H. et al., Nature, 451: 141-146 (2008)

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a means of improvingthe efficiency of establishment of iPS cells; another object of thepresent invention is to provide a method of efficiently producing iPScells using the means.

The present inventors conducted extensive investigations with the aim ofaccomplishing the above-described objects, and found that by inhibitingthe p53 function in the step of somatic cell nuclear reprogramming, theefficiency of establishment of iPS cells can be remarkably increased.The effect was particularly remarkable in human cells. Also, byinhibiting the p53 function, even with 3 factors, an establishmentefficiency closer to the efficiency with 4 factors was obtained, than bya conventional method. Furthermore, the present inventors succeeded inestablishing iPS cells with ease by deleting the p53 function even for Tlymphocytes, for which it has conventionally been thought to bedifficult to establish iPS cells, and have developed the presentinvention.

Accordingly, the present invention provides:

-   [1] A method of improving the efficiency of establishment of iPS    cells, comprising inhibiting the p53 function in the step of somatic    cell nuclear reprogramming.

[2] The method according to [1] above, wherein the p53 function isinhibited by bringing a chemical inhibitor of p53 into contact with asomatic cell.

[3] The method according to [1] above, wherein the p53 function isinhibited by bringing a dominant negative mutant of p53 or a nucleicacid that encodes the same into contact with a somatic cell.

[4] The method according to [1] above, wherein the p53 function isinhibited by bringing a nucleic acid selected from the group consistingof siRNAs and shRNAs against p53 and DNAs that encode the same intocontact with a somatic cell.

[5] The method according to [1] above, wherein the p53 function isinhibited by bringing an inhibitor of p53 pathway into contact with asomatic cell.

[6] An agent for improving the efficiency of establishment of iPS cells,the agent comprising an inhibitor of p53 function.

[7] The agent according to [6] above, wherein the inhibitor is achemical inhibitor.

[8] The agent according to [6] above, wherein the inhibitor is adominant negative mutant of p53 or a nucleic acid that encodes the same.

[9] The agent according to [6] above, wherein the inhibitor is a nucleicacid selected from the group consisting of siRNAs and shRNAs against p53and DNAs that encode the same.

[10] The agent according to [6] above, wherein the inhibitor is aninhibitor of p53 pathway.

[11] A method of producing iPS cells, comprising bringing a nuclearreprogramming substance and an inhibitor of p53 functional into contactwith a somatic cell.

[12] The method according to [11] above, wherein the inhibitor is achemical inhibitor.

[13] The method according to [11] above, wherein the inhibitor is adominant negative mutant of p53 or a nucleic acid that encodes the same.

[14] The method according to [11] above, wherein the inhibitor is anucleic acid selected from the group consisting of siRNAs and shRNAsagainst p53 and DNAs that encode the same.

[15] The method according to [11] above, wherein the inhibitor is aninhibitor of p53 pathway.

[16] The method according to [11] above, wherein the nuclearreprogramming substances are Oct3/4, Klf4 and Sox2, or nucleic acidsthat encode the same.

[17] The method according to [11] above, wherein the nuclearreprogramming substances are Oct3/4, Klf4, Sox2 and c-Myc, or nucleicacids that encode the same.

[18] The method according to [11] above, wherein the somatic cell is a Tcell.

[19] An iPS cell wherein a T cell antigen receptor (TCR) gene isrearranged, the iPS cell being obtained by reprogramming a T cell.

[20] An iPS cell comprising a dominant negative mutant of p53 or anexogenous nucleic acid that encodes an siRNA or shRNA against p53.

Because inhibitors of p53 function make it possible to remarkablyincrease the efficiency of establishment of iPS cells, the same areparticularly useful in the induction of iPS cells by means of 3 factorsexcept c-Myc, for which the efficiency of establishment hasconventionally been very low. Because c-Myc is feared to causetumorigenesis when reactivated, the improvement in the efficiency ofestablishment of iPS cells using the 3 factors is of paramount utilityin applying iPS cells to regenerative medicine.

Because the iPS cells derived from a finally differentiated T cell haveTCR already rearranged therein, the iPS cells are useful as a T cellimmunotherapeutic agent, provided that the iPS cells are induced from aT cell that recognizes a cell that presents a particular antigen (e.g.,cancer cells, infected cells and the like), amplified in large amounts,and allowed to re-differentiate into cytotoxic T cells (CTLs).

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows results of an examination of effects of p53 deficiency onthe establishment of iPS cells. FIG. 1(A) and FIG. 1(B) show results ofintroduction of 4 factors (Oct3/4, Sox2, Klf4, c-Myc) to induce iPScells, and results of introduction of 3 factors (Oct3/4, Sox2, Klf4) toinduce iPS cells, respectively. In the figures, “p53+/−” shows resultsfor p53-hetero-deficient cells (control); “p53−/−” shows results forp53-homo-deficient cells. In the figures, the axis of ordinatesindicates the number of GFP-positive colonies. Each graph shows a totalof three experiments.

FIG. 2 is a photograph showing that a GFP-positive colony resulting fromp53-homo-deficient cells infected with 4 factors (Oct3/4, Sox2, Klf4,c-Myc) was subcutaneously injected to an immunodeficient mouse, and thatteratomas were formed.

FIG. 3 (A)-(D) show results of an examination of effects of introductionof a dominant negative mutant of p53 (p53P275S) on the establishment ofiPS cells. FIG. 3(A) shows an outline of the experimental procedure.FIG. 3(B) and FIG. 3(C) show results of introduction of 4 factors andresults of introduction of 3 factors, respectively. In the figures,“P275S” shows results of introduction of p53P275S. In the figures,“DsRed” and “p53” show results of introduction of DsRedExpress andmutation-free wild-type p53, respectively, in place of p53P275S. Theaxis of ordinates indicates the number of GFP-positive colonies. FIG.3(D) shows photographs of colonies corresponding to the respectiveresults.

FIG. 3 (E)-(G) show results of an examination of effects of introductionof p53 into p53-homo-deficient mice on the establishment of iPS cells.FIG. 3(E) and FIG. 3 (F) show results of introduction of 4 factors and 3factors, respectively. In the figures, “DsRed” shows results ofintroduction of DsRedExpress; “p53” shows results of introduction ofwild-type p53. The axis of ordinates indicates the number ofGFP-positive colonies. FIG. 3(G) shows photographs of coloniescorresponding to the respective results.

FIG. 4 shows results of an examination of effects of treatment withPifithrin, a p53 inhibitor, on the establishment of iPS cells. FIG. 4(A)shows an outline of the experimental procedure; FIG. 4(B) showsexperimental results. In FIG. 4(B), “DMSO” shows results of treatmentwith DMSO (control); “Pifithrin α, p-cyclic, nitro” shows results oftreatment with Pifithrin. The axis of ordinates indicates the number ofGFP-positive colonies.

FIG. 5 is a photographic representation showing that ES-like cellsresulting from T cells derived from a Nanog-GFP/Trp53^(−/−) mouseinfected with 4 factors (Oct3/4, Sox2, Klf4, c-Myc) are GFP-positive.Left panel: phase-contrast image, right panel: GFP-positive colonyimage. In the figure, 408E2, 408E7, and 408E8 indicate clone numbers.

FIG. 6 is a photographic representation of RT-PCR showing that ES-likecells resulting from T cells derived from a Nanog-GFP/Trp53^(−/−) mouseinfected with 4 factors (Oct3/4, Sox2, Klf4, c-Myc) expressES-cell-specific genes. In the figure, Oct3/4 to Zfp296 are ES cellmarkers, and FasL to Ifng are T cell markers. Natl and Trim28 arepositive controls, and Oct3/4 Tg to c-Myc Tg confirm the expression ofthe 4 factors introduced. In the figure, “CD90⁺T” and “Spleen” indicatethe T cells and spleen that served as cell sources for iPS cellinduction, respectively; 7B3 and 38D2 indicate Fbx15 iPS cells (Nature448, 313-317 (2007)) and Nanog iPS cells (Nature 448, 313-317 (2007)),respectively.

FIG. 7 is a photographic representation showing that ES-like cellsresulting from T cells derived from a Nanog-GFP/Trp53 mouse infectedwith 4 factors (Oct3/4, Sox2, Klf4, c-Myc) are positive for ES cellmarkers SSEA1 and alkaline phosphatase.

FIG. 8 is a photographic representation showing results confirming thatES-like cells resulting from T cells derived from aNanog-GFP/Trp53^(−/−) mouse infected with 4 factors (Oct3/4, Sox2, Klf4,c-Myc) possess a potential for differentiating into three germ layers,by staining using AFP, GATA4, α-SMA, Desmin, βIII-tubulin and GFAPantibodies.

FIG. 9 is a graphic representation of results of a DNA microarrayanalysis performed to determine whether there is a difference inexpression pattern between MEF isolated from a p53-homo-deficient mouseand MEF isolated from an ordinary, non-p53-deficient mouse. (A) Allgenes were detected, (B) genes expressed specifically in ES cells onlywere detected, (C) genes expressed specifically in fibroblasts (MEF)only were detected.

FIG. 10 shows an adult chimeric mouse resulting from an ES-like cellderived from a T cell of a Nanog-GFP/Trp53^(−/−) mouse infected with 4factors (Oct3/4, Sox2, Klf4, c-Myc).

FIG. 11 is a photographic representation showing results confirming therearrangement of the V-(D)-J DNA of the Tcrβ gene by genomic PCR. In thefigure, “GL” indicates a germline band.

FIG. 12 shows iPS generation from p53-null MEF by the four or threefactors. (a) iPS cells were generated from Nanog-GFP reporter MEF, whichwere either p53 wild-type, heterozygous, or homozygous, by the threefactors. After retroviral transduction, 5000 live cells were collectedby a flowcytometer. GFP-positive colonies were counted 28 days after thetransduction and shown of the top of the graphs. Data of threeindependent experiments are shown. (b) iPS cells were generated by thethree factors from single sorted cells in wells of 96-well plates.GFP-positive colonies were counted 28 days after the transduction. Datafrom three independent experiments are shown. (c) iPS cells weregenerated by the four factors, including c-Myc, from single sorted cellsin wells of 96-well plates. GFP-positive colonies were counted 21 daysafter the transduction. Data from three independent experiments areshown.

FIG. 13 shows iPS generation from p53 heterozygous or homozygous MEFs bythe three factors co-transduced with wild-type or mutant p53. (a)Retrovirus expressing either the dominant negative p53 mutant (P275S) orwild-type was co-transduced with the three factors into Oct3/4-GFP, p53heterozyous MEFs. After retroviral transduction, 5000 cells werecollected and GFP-positive colonies were counted 28 days after thetransduction. Data of three independent experiments are shown. (b)Retrovirus expressing either the wild-type or mutant p53 wasco-tranduced with the three factors into Nanog-GFP, p53 homozyous MEFs.After retroviral transduction, 5000 live cells were collected andGFP-positive colonies were counted 28 days after the transduction. Dataof two independent experiments are shown.

FIGS. 14-16 show characterization of iPS cells derived from p53hetrozygous or homozygous MEFs.

FIG. 14 shows phase contrast images (upper) and fluorescent images(lower) of iPS cells derived from Nanog-GFP, p53-null MEFs by the threeor four factors. Bars indicate 100

FIG. 15 shows RT-PCR analysis of the expression of ES cell marker genes,p53 and the four factors. By using specific sets of primers, the totalexpression, endogenous expression and transgene expression of the fourfactors were distinguished.

FIG. 16 shows histological examination of teratomas derived fromp53-null iPS cells with the three (a) or four (b) factors. (a) Shown arehematoxylin-eosin staining of neural tissues (upper left), cartilage(upper right), muscle (lower left), and gut-like epithelial tissues(lower right). (b) Shown are hematoxylin-eosin staining ofundifferentiated cells (upper) and neural tissues (lower).

FIGS. 17-20 show increased efficiency of human iPS cell generation byp53 suppression.

FIG. 17 shows effects of mutant p53 co-transduction on iPS generationfrom HDFs by the four or three factors. The retroviral vector expressingeither P275S or DD was transduced into HDFs together with the four orthree reprogramming factors. Shown are the numbers of iPS cell coloniesby the four factors ((a), from 5×10³ HDFs) and by the three factors((b), from 4×10⁴ HDFs). FIG. 17 c shows teratomas derived from human iPScells, which were generated with the three reprogramming factors and thep53DD mutant. Shown are hematoxylin-eosin staining of neural tissues(upper left), cartilage (upper right), muscle (lower left), and gut-likeepithelial tissues (lower right).

FIG. 18 shows suppression of p53 production by p53 shRNA. Retroviralvectors for p53 shRNA or control RNA were transduced into HDFs. Six daysafter the transduction, p53 protein levels were determined by westernblot analyses.

FIG. 19 shows effects of p53 shRNA co-transduction on iPS generationfrom HDFs by the four factors. The retroviral vector expressing eitherp53 shRNA or control RNA was transduced into HDFs together with the fourreprogramming factors. To rescue RNAi-mediated knockdown, a retroviralvector for mouse p53 was co-introduced. Shown are the numbers of iPScolonies in four experiments.

FIG. 20 shows effects of p53 shRNA co-transduction on iPS generationfrom HDFs by the three factors. The retroviral vector expressing eitherp53 shRNA or control RNA was transduced into HDFs together with thethree reprogramming factors. To rescue RNAi-mediated knockdown, aretroviral vector for mouse p53 was co-introduced. Shown are the numbersof iPS colonies from 5×10⁴ HDFs (a) or 5×10⁵ HDFs (b) in twoexperiments.

FIG. 21 shows effects of MDM2 co-transduction on iPS generation fromHDFs by the four or three reprogramming factors. The retroviral vectorexpressing MDM2, p53 shRNA or RB shRNA, or control vector was transducedinto HDFs together with the four factors (a) or three factors (b). Shownare the numbers of iPS colonies from 5×10⁴ cells of HDF.

FIG. 22 shows photographs demonstrating the expression ofendoderm-(AFP), mesoderm-(α-SMA) and ectoderm-(βIII-tubulin)differentiation markers in the cells that differentiated from the iPSclones.

FIG. 23 shows marker gene expressions in undifferentiated cells (U) andin differentiated cells after embryoid body formation (D). “Mock” showsco-transduction of empty vector (pMKO.1-puro) with the threereprogramming factors. “p53 shRNA-2” shows co-transduction of p53shRNA-2 with the three reprogramming factors.

FIG. 24 shows effects of p53 suppression on p21 and Myc. Genes regulated(4 increased and 7 decreased) by p53 suppression were introduced intoHDFs together with the four reprogramming factors (a) or the fourreprogramming factors and the p53 shRNA (b). On day 24 (a) or day 28 (b)post-transduction, numbers of iPS cell colonies were counted. **; p<0.01compared to DsRed control (n=3). Luciferase reporters containingresponsive elements of p53 or Myc, or that driven by the polymerase IIpromoter were introduced into HDFs, together with the mock retroviralvector, the p53 shRNA, the four reprogramming factors, or the threefactors devoid of Myc. Two days later, luciferase activities weredetermined (c). **; p<0.01, *; p<0.05 compared to the mock control(n=3).

FIG. 25 a shows an outline of the experimental procedure. FIG. 25 bshows results of introduction of 4 factors (Oct3/4, Sox2, Klf4, c-Myc).In the figures, “+/+” shows results for wild-type cells (control); “−/−”shows results for p53-homo-deficient cells. In the figures, the axis ofordinates indicates the number of GFP-positive colonies. FIG. 25 c showsresults of examinations of integration of plasmid DNAs into the genome(upper panel: genomic PCR; lower panel: Southern blot analysis). FIG. 25d shows photographs of the obtained cells (upper left: phase-contrastimage, upper right: GFP-positive colony image, lower left: merge ofphase-contrast image and GFP-positive colony image) and a chimeric mouseresulting from an ES-like cell obtained (lower right panel).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of improving the efficiency ofestablishment of iPS cells by inhibiting the p53 function in the step ofsomatic cell nuclear reprogramming. The choice of means of inhibitingthe p53 function is not particularly limited; preferably, a methodwherein an inhibitor of p53 function is brought into contact with asomatic cell can be mentioned.

As mentioned herein, “an inhibitor of p53 function” may be anysubstance, as far as it is capable of inhibiting either (a) the functionof the p53 protein or (b) the expression of the p53 gene. That is, notonly substances that act directly on the p53 protein to inhibit thefunction thereof and substances that act directly on the p53 gene toinhibit the expression thereof, but also substances that act on a factorinvolved in p53 signal transduction to result in inhibition of thefunction of the p53 protein or the expression of the p53 gene, are alsoincluded in the scope of “an inhibitor of p53 function” as mentionedherein.

Examples of substances that inhibit the function of the p53 proteininclude, but are not limited to, a chemical inhibitor of p53, a dominantnegative mutant of p53 or a nucleic acid that encodes the same, ananti-p53 antagonist antibody or a nucleic acid that encodes the same, adecoy nucleic acid comprising a consensus sequence of a p53 responsiveelement, a substance that inhibits the p53 pathway, and the like.Preferably, a chemical inhibitor of p53, a dominant negative mutant ofp53 or a nucleic acid that encodes the same, and a p53 pathway inhibitorcan be mentioned.

(a1) Chemical Inhibitors of p53

Examples of chemical inhibitors of p53 include, but are not limited to,p53 inhibitors typified by pifithrin (PFT)-α and -β, which are disclosedin WO 00/44364, PFT-μ disclosed in Storm et al. (Nat. Chem. Biol. 2, 474(2006)), analogue thereof and salts thereof (for example, acid additionsalts such as hydrochlorides and hydrobromides, and the like) and thelike. Thereof, PFT-α and analogues thereof[2-(2-Imino-4,5,6,7-tetrahydrobenzothiazol-3-yl)-1-p-tolylethanone, HBr(product name: Pifithrin-α) and1-(4-Nitrophenyl)-2-(4,5,6,7-tetrahydro-2-imino-3(2H)-benzothiazolyl)ethanone,HBr (product name: Pifithrin-α, p-Nitro)], PFT-β and analogues thereof[2-(4-Methylphenyl)imidazo[2,1-b]-5,6,7,8-tetrahydrobenzothiazole, HBr(product name: Pifithrin-α, Cyclic) and2-(4-Nitrophenyl)imidazo[2,1-b]-5,6,7,8-tetrahydrobenzothiazole (productname: Pifithrin-α, p-Nitro, Cyclic)], and PFT-μ[Phenylacetylenylsulfonamide (product name: Pifithrin-μ)] arecommercially available from Merck.

Contact of a chemical inhibitor of p53 with a somatic cell can beperformed by dissolving the inhibitor at an appropriate concentration inan aqueous or non-aqueous solvent, adding the solution of the inhibitorto a medium suitable for cultivation of somatic cells isolated from ahuman or mouse (for example, minimal essential medium (MEM), Dulbecco'smodified Eagle medium (DMEM), RPMI1640 medium, 199 medium, F12 mediumand the like supplemented with about 5 to 20% fetal bovine serum) sothat the inhibitor concentration will fall in a range that fullyinhibits the p53 function and does not cause cytotoxicity, and culturingthe cells for a given period. The inhibitor concentration variesdepending on the kind of inhibitor used, and is chosen as appropriateover the range of about 0.1 nM to about 100 nM. Duration of contact isnot particularly limited, as far as it is sufficient to achieve nuclearreprogramming of the cells; usually, the inhibitor may be allowed toco-present in the medium until a pluripotent marker positive colonyemerges.

The p53 gene is known as a tumor suppressor gene; permanent inhibitionof p53 function potentially increases the risk of carcinogenesis.Chemical inhibitors of p53 are extremely useful, not only because of theadvantage of permitting introduction into cells simply by the additionto the medium, but also because of the ability to terminate theinhibition of p53 function, easily and quickly, by removing the mediumcontaining the inhibitor after induction of iPS cells.

(a2) Dominant Negative Mutants of p53

The choice of dominant negative mutant of p53 is not particularlylimited, as far as the mutant is capable of competitively acting againstthe wild-type p53 protein endogenously expressed in somatic cells toinhibit the function thereof; for example, p53P275S, resulting frompoint mutation of the proline at the position 275 (in the case ofhumans, position 278) located in the DNA-binding region of mouse p53 toserine (de Vries, A., Proc. Natl. Acad. Sci. USA, 99, 2948-2953 (2002));p53DD, resulting from deletion of the amino acids at the positions14-301 of mouse p53 (in human p53, corresponds to the positions 11-304)(Bowman, T., Genes Develop., 10, 826-835 (1996)), and the like can bementioned. Other known mutants include, for example, p53S58A, resultingfrom point mutation of the serine at the position 58 of mouse p53 (inthe case of humans, position 61) to alanine; p53C135Y, resulting frompoint mutation of the cysteine at the position 135 of human p53 (in thecase of mice, position 132) to tyrosine; p53A135V, resulting from pointmutation of the alanine at the position 135 of mouse p53 (in the case ofhumans, position 138) to valine; p53R172H, resulting from point mutationof the arginine at the position 172 (in the case of humans, position175) to histidine; p53R270H, resulting from point mutation of thearginine at the position 270 (in the case of humans, position 273) tohistidine; p53D278N, resulting from point mutation of the aspartic acidat the position 278 of mouse p53 (in the case of humans, position 281)to asparagine, and the like; these can be used in the same way.

A dominant negative mutant of p53 can be obtained by for example, thetechnique described below. First, an appropriate oligonucleotide issynthesized as a probe or primer on the basis of the mouse or human p53cDNA sequence information shown by SEQ ID NO:1 or 3, and a mouse orhuman p53 cDNA is cloned from a mRNA, cDNA or cDNA library derived froma mouse or human cell or tissue, using the hybridization method or the(RT-)PCR method, and is subcloned into an appropriate plasmid. In a formwherein a codon of the site into which a mutation is to be introduced(for example, in the case of p53P275S, cct, which is shown by nucleotidenumbers 951-953 in the nucleotide sequence shown by SEQ ID NO:1) isreplaced with a codon that encodes another desired amino acid (forexample, in the case of p53P275S, tct), a primer comprising the site issynthesized, and inverse PCR is performed using this primer with theplasmid incorporating the p53 cDNA as a template, whereby a nucleic acidthat encodes the desired dominant negative mutant is acquired. In thecase of a deletion mutant like p53DD, a primer may be designed outsidethe site to be deleted, and inverse PCR may be performed as describedabove. By introducing the thus-obtained nucleic acid that encodes thedominant negative mutant into a host cell, and recovering a recombinantprotein from the cultured cell or its conditioned medium, the desireddominant negative mutant can be acquired.

Contact of a dominant negative mutant with a somatic cell can beachieved using a method known per se for protein transfer into a cell.Such methods include, for example, the method using a protein transferreagent, the method using a protein transfer domain (PTD)- or cellpenetrating peptide (CPP)-fusion protein, the microinjection method andthe like. Protein transfer reagents are commercially available,including BioPOTER Protein Delivery Reagent (Gene Therapy Systems),Pro-Ject™ Protein Transfection Reagent (PIERCE) and ProVectin (IMGENEX),which are based on a cationic lipid; Profect-1 (Targeting Systems),which is based on a lipid; Penetrain Peptide (Q biogene) and Chariot Kit(Active Motif), which are based on a membrane-permeable peptide, andGenomONE (Ishihara Sangyo), which is based on HVJ envelop (inactivatedSendai virus), and the like. The transfer can be achieved per theprotocols attached to these reagents, a common procedure being asdescribed below. A dominant negative mutant of p53 is diluted in anappropriate solvent (for example, a buffer solution such as PBS orHEPES), a transfer reagent is added, the mixture is incubated at roomtemperature for about 5 to 15 minutes to form a complex, this complex isadded to the cells after medium exchange with a serum-free medium, andthe cells are incubated at 37° C. for one to several hours. Thereafter,the medium is removed and replaced with a serum-containing medium.

Developed PTDs include those using the cell penetrating domain of aprotein, such as drosophila-derived AntP, HIV-derived TAT (Frankel, A.et al, Cell 55, 1189-93 (1988) or Green, M. & Loewenstein, P. M. Cell55, 1179-88 (1988)), Penetratin (Derossi, D. et al, J. Biol. Chem. 269,10444-50 (1994)), Buforin II (Park, C. B. et al. Proc. Natl Acad. Sci.USA 97, 8245-50 (2000)), Transportan (Pooga, M. et al. FASEB J. 12,67-77 (1998)), MAP (model amphipathic peptide) (Oehlke, J. et al.Biochim. Biophys. Acta. 1414, 127-39 (1998)), K-FGF (Lin, Y. Z. et al.J. Biol. Chem. 270, 14255-14258 (1995)), Ku70 (Sawada, M. et al. NatureCell Biol. 5, 352-7 (2003)), Prion (Lundberg, P. et al. Biochem.Biophys. Res. Commun. 299, 85-90 (2002)), pVEC (Elmquist, A. et al. Exp.Cell Res. 269, 237-44 (2001)), Pep-1 (Morris, M. C. et al. NatureBiotechnol. 19, 1173-6 (2001)), Pep-7 (Gao, C. et al. Bioorg. Med. Chem.10, 4057-65 (2002)), SynB1 (Rousselle, C. et al. MoI. Pharmacol. 57,679-86 (2000)), HN-I (Hong, F. D. & Clayman, G L. Cancer Res. 60, 6551-6(2000)), and HSV-derived VP22. CPPs derived from the PTDs includepolyarginines such as 11R (Cell Stem Cell, 4:381-384 (2009)) and 9R(Cell Stem Cell, doi:10.1016/j.stem.2009.05.005 (2009)). A fusionprotein expression vector incorporating a cDNA of a dominant negativemutant of p53 and a PTD or CPP sequence is prepared to allow recombinantexpression of the fusion protein, and the fusion protein is recoveredfor use in the transfer. This transfer can be achieved as describedabove, except that no protein transfer reagent is added.

Microinjection, a method of placing a protein solution in a glass needlehaving a tip diameter of about 1 μm, and injecting the solution into acell, ensures the transfer of the protein into the cell.

As described above, permanent inhibition of p53 function potentiallyincreases the risk of carcinogenesis; however, because a dominantnegative mutant of p53 undergoes degradation by protease in thetransfected cell and disappears gradually, and correspondingly the p53function endogenously occurring in the cell is restored, use of themutant protein can be suitable in cases where high safety is required asin the case where the iPS cells obtained are utilized for therapeuticpurposes.

(a3) Nucleic Acids that Encode a Dominant Negative Mutant of p53

However, taking into account the ease of introduction into a somaticcell, a dominant negative mutant of p53 may be used in the form of anucleic acid that encodes a protein, rather than of the protein itself.Therefore, in another preferred mode of embodiment of the presentinvention, the inhibitor of p53 function is a nucleic acid that encodesa dominant negative mutant of p53. The nucleic acid may be a DNA or anRNA, or a DNA/RNA chimera, and is preferably a DNA. The nucleic acid maybe double-stranded or single-stranded. A cDNA that encodes a dominantnegative mutant of p53 can be cloned by the technique described abovewith respect to preparation of the mutant protein.

The cDNA isolated is inserted into an appropriate expression vectorcomprising a promoter capable of functioning in a target somatic cell.Useful expression vectors include, for example, viral vectors such asretrovirus, lentivirus, adenovirus, adeno-associated virus, herpesvirusand Sendai virus, plasmids for the expression in animal cells (e.g.,pA1-11, pXT1, pRc/CMV, pRc/RSV, pcDNAI/Neo) and the like. A kind ofvector used can be chosen as appropriate according to the intended useof the iPS cells obtained.

Useful promoters used in the expression vector include, for example, SRαpromoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter,RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney mouse leukemia virus)LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter, EF-alphapromoter, CAG promoter and the like. Preference is given to MoMuLV LTR,CMV promoter, SRα promoter, EF-alpha promoter, CAG promoter and thelike.

The expression vector may harbor, as desired, in addition to a promoter,an enhancer, a polyadenylation signal, a selectable marker gene, an SV40replication origin and the like. Examples of the selectable marker geneinclude the dihydrofolate reductase gene, the neomycin resistance gene,the puromycin resistance gene and the like.

An expression vector harboring a nucleic acid encoding a m dominantnegative mutant of p53 can be introduced into a cell by a techniqueknown per se according to the kind of the vector. In the case of a viralvector, for example, a plasmid containing the nucleic acid encoding adominant negative mutant of p53 is introduced into an appropriatepackaging cell (e.g., Plat-E cells) or a complementary cell line (e.g.,293-cells), the viral vector produced in the culture supernatant isrecovered, and the vector is infected to the cell by a method suitablefor the viral vector. For example, specific means using a retroviralvector as a vector are disclosed in WO2007/69666, Cell, 126, 663-676(2006) and Cell, 131, 861-872 (2007); when a lentiviral vector is usedas a vector, a disclosure is available in Science, 318, 1917-1920(2007). When iPS cells are utilized for therapeutic purposes, permanentinhibition of the p53 function potentially increases the risk ofcarcinogenesis in tissues and organs differentiated from iPS cells;therefore, the nucleic acid that encodes a dominant negative mutant ofp53 is preferably expressed transiently, without being integrated intothe chromosome of the cells. From this viewpoint, use of an adenoviralvector, whose integration into chromosome is rare, is preferred.Specific means using an adenoviral vector is disclosed in Science, 322,945-949 (2008). Because adeno-associated virus is also low in thefrequency of integration into chromosome, and is lower than adenoviralvectors in terms of cytotoxicity and inflammation-causing activity, itcan be mentioned as another preferred vector. Because persistentexpression type Sendai viral vector is capable of being stably presentoutside the chromosome, and can be degraded and removed using an siRNAas required, it is preferably utilized as well. Regarding persistentexpression type Sendai viral vector, one described in J. Biol. Chem.,282, 27383-27391 (2007) can be used.

When a retroviral vector or a lentiviral vector is used, even ifsilencing of the transgene has occurred, it possibly becomesreactivated; therefore, for example, a method can be used preferablywherein a nucleic acid that encodes a dominant negative mutant of p53 iscut out using the Cre/loxP system, when becoming unnecessary. That is,with a loxP sequence arranged on both ends of the nucleic acid inadvance, iPS cells are induced, thereafter the Cre recombinase isallowed to act on the cells using a plasmid vector or adenoviral vector,and the region sandwiched by the loxP sequences can be cut out. Becausethe enhancer-promoter sequence of the LTR U3 region possibly upregulatesa host gene in the vicinity thereof by insertion mutation, it is morepreferable to avoid the expression regulation of the endogenous gene bythe LTR outside of the loxP sequence remaining in the genome withoutbeing cut out, using a 3′-self-inactivated (SIN) LTR prepared bydeleting the sequence, or substituting the sequence with apolyadenylation sequence such as of SV40. Specific means using theCre-loxP system and SIN LTR is disclosed in Chang et al., Stem Cells,27: 1042-1049 (2009).

Meanwhile, in the case of a plasmid vector, which is a non-viral vector,the vector can be introduced into a cell using the lipofection method,liposome method, electroporation method, calcium phosphateco-precipitation method, DEAE dextran method, microinjection method,gene gun method and the like. Also when a plasmid vector is used, itsintegration into chromosome is rare, the transgene is degraded andremoved by DNase in the cells; therefore, when iPS cells are utilizedfor therapeutic purposes, use of a plasmid vector can be a preferredmode of embodiment. A specific means using a plasmid as a vector isdescribed in, for example, Science, 322, 949-953 (2008) and the like.

Another preferable non-integration type vector is an episomal vector,which is autonomously replicable outside chromosome. Specific meansusing an episomal vector is disclosed in Science, 324, 797-801 (2009).

Also when an adenovirus or a plasmid is used, the transgene can getintegrated into chromosome; therefore, it is eventually necessary toconfirm the absence of insertion of the gene into chromosome by Southernblotting or PCR. For this reason, like the aforementioned Cre-loxPsystem, it can be advantageous to use a means wherein the transgene isintegrated into chromosome, thereafter the gene is removed. In anotherpreferred mode of embodiment, a method can be used wherein the transgeneis integrated into chromosome using a transposon, thereafter atransferase is allowed to act on the cell using a plasmid vector oradenoviral vector so as to completely eliminate the transgene from thechromosome. As examples of preferable transposons, piggyBac, atransposon derived from a lepidopterous insect, and the like can bementioned. Specific means using the piggyBac transposon is disclosed inKaji et al., Nature advance online publication 1 Mar. 2009(doi:10.1038/nature07864), Woltjen et al., Nature advance onlinepublication 1 Mar. 2009 (doi:10.1038/nature07863). In anotherembodiment, tetracycline responsive element in promoter region (Tet-On®& Tet-Off® Gene Expression Systems, Clontech) can be used for theexcision of transgenes.

(a4) p53 Pathway Inhibitors

Here, the term p53 pathway is used with a meaning including all upstreamsignal cascades that can activate p53 and all downstream signal cascadesmediated by activated p53. Therefore, p53 pathway inhibitors include allsubstances that inhibit any one of the aforementioned signaltransduction pathways, but in a preferred mode of embodiment, the p53pathway inhibitor is a substance that inhibits the expression orfunction (Myc inhibitory activity) of p21, whose transcription isactivated by p53; for example, siRNA, shRNA, antisense nucleic acids,ribozymes against p21 and the like can be mentioned. These nucleic acidsthat inhibit the expression of p21 can be designed and synthesized inthe same manner as the method for siRNA, shRNA, antisense nucleic acids,and ribozymes against p53 described below, and can be introduced into asomatic cell. The nucleic acids may be provided in the form of a vectorthat expresses them, the vector can be constructed in the same manner asthe method for a vector that expresses an siRNA, shRNA, antisensenucleic acid, or ribozyme against p53 described below, and introducedinto a somatic cell.

In another preferred mode of embodiment, the p53 pathway inhibitor is asubstance that inhibits the ARF-MDM2-p53 pathway; for example, asARF-MDM2-p53 pathway inhibitors, MDM2, which binds directly to p53 topromote the extranuclear transportation or ubiquitination thereof or anucleic acid that encodes the same, p19^(ARF), which inhibits the actionof MDM2 on p53, a substance that inhibits the expression or function ofATM (ataxia-telangiectasia mutated) (for example, siRNAs and shRNAsagainst these factors) and the like can be mentioned.

(a5) Other Substances

As examples of other substances that inhibit the function of the p53protein, anti-p53 antagonist antibody or a nucleic acid that encodes thesame can be mentioned. The anti-p53 antagonist antibody may be apolyclonal antibody or a monoclonal antibody. The isotype of theantibody is not particularly limited, and is preferably IgG, IgM or IgA,particularly preferably IgG. The antibody may be, in addition to acomplete antibody molecule, for example, a fragment such as Fab, Fab′,or F(ab′)₂, a conjugate molecule prepared by a gene engineeringtechnique, such as scFv, scFv-Fc, minibody, or diabody, or a derivativethereof modified with a molecule having protein-stabilizing action, suchas polyethylene glycol (PEG). An anti-p53 antagonist antibody can beproduced using p53 or a partial peptide thereof as an antigen, by amethod of antibody or anti-serum production known per se. As examples ofknown anti-p53 antagonist antibodies, PAb1801 (Oncogene Science Ab-2)and DO-1 (Oncogene Science Ab-6) (Gire and Wynford-Thomas, Mol. Cell.Biol., 18, 1611-1621 (1998)) and the like can be mentioned. A nucleicacid that encodes an anti-p53 antagonist antibody can be isolated from ahybridoma that produces an anti-p53 monoclonal antibody by aconventional method. The H-chain and L-chain genes obtained may bejoined together to prepare a nucleic acid that encodes a single-chainantibody. Preferably, these antibodies are fused with aforementioned PTDor CPP.

As another substance that inhibits the function of the p53 protein, ananti-p21 antagonist antibody or a nucleic acid that encodes the same canbe mentioned. An anti-p21 antagonist antibody and a nucleic acid thatencodes the same can also be prepared as with the aforementionedanti-p53 antagonist antibody and nucleic acid that encodes the same.

Still another substance that inhibits the function of the p53 protein isa decoy nucleic acid comprising a consensus sequence of p53-responsiveelement (e.g., Pu-Pu-Pu-G-A/T-T/A-C-Py-Py-Py (Pu: purine base, Py:pyrimidine base); SEQ ID NO:27). Such a nucleic acid can be synthesizedon the basis of the aforementioned nucleotide sequence information usingan automated DNA/RNA synthesizer. Alternatively, such a decoy nucleicacid is commercially available (e.g., p53 transcription factor decoy(GeneDetect.com)).

An anti-p53 antagonist antibody and an anti-p21 antagonist antibody, ora nucleic acid that encodes the antibody can be introduced into a cellwith the method described in the statement of a dominant negative mutantof p53 or a nucleic acid that encodes the mutant, respectively. Theaforementioned decoy nucleic acid can be introduced into a cell bylipofection method and the like.

Meanwhile, as examples of substances that inhibit the expression of thep53 gene, siRNAs or shRNAs against p53, vectors that express an siRNA orshRNA against p53, antisense nucleic acids against p53 and ribozymesagainst p53, and the like can be mentioned, and siRNAs and shRNAsagainst p53 and vectors that express an siRNA or an shRNA arepreferable.

(b1) siRNA and shRNA Against p53

An siRNA against p53 can be designed on the basis of the mouse or humanp53 cDNA sequence information shown by SEQ ID NO:1 or 3, in accordancewith, for example, the rules proposed by Elbashir et al. (Genes Dev.,15, 188-200 (2001)). The target sequence for the siRNA is, as a generalrule, AA+(N)₁₉, but may be AA+(N)₂₁ or NA+(N)₂₁. The 5′ end of the sensestrand need not to be AA. Although the position of the target sequenceis not particularly limited, it is desirable that the target sequence beselected between 5′-UTR and about 50 bases from the start codon, as wellas from a region other than 3′-UTR. The GC content of the targetsequence is also not particularly limited, but the content is preferablyabout 30 to about 50%; a sequence with no irregularity in GCdistribution and with only a few repeats is desirable. When a polIIIpromoter is used as a promoter in designing a vector that expresses ansiRNA or shRNA of (b2) below, a sequence of 4 or more T or A bases insuccession should not be chosen, so as to prevent polymerasetranscription from ceasing.

The target sequence candidates selected on the basis of theabove-described rules are examined for homology to sequences of 16-17bases in succession in mRNAs other than the target, using a homologysearch software program such as BLAST(http://www.ncbi.nlm.nih.gov/BLAST/), so as to confirm the specificityof the target sequences selected. For the target sequences for which thespecificity has been confirmed, a double-stranded RNA consisting of asense strand having a 3′-terminal overhang of TT or UU in 19-21 basesafter AA (or NA) and a sequence complementary to the 19-21 bases, and anantisense strand having a 3′-terminal overhang of TT or UU, is designedas an siRNA. Also, an shRNA can be designed by choosing as appropriatean optionally chosen linker sequence capable of forming a loop structure(for example, about 8-25 bases), and ligating the aforementioned sensestrand and antisense strand via the linker sequence.

Sequences of siRNAs and/or shRNAs can be searched for using searchsoftware programs available at no cost on various websites. Examples ofsuch sites include, but are not limited to, the siRNA Target Finder(http://www.ambion.com/jp/techlib/misc/siRNA_finder.html) and insertdesign tool for pSilencer™ Expression Vector(http://www.ambion.com/jp/techlib/misc/psilencer_converter.html), bothprovided by Ambion, and GeneSeer(http://codex.cshl.edu/scripts/newsearchhairpin.cgi), provided by RNAiCodex; and similar search is possible on the websites of QIAGEN, TakaraBio, SiSearch, Dharmacon, Whitehead Institute, Invitrogen, Promega andthe like.

Shown below are the sequences of shRNAs against mouse p53 designed usingsoftware programs available on the websites of Ambion (SEQ ID NO:5-24)and RNAi Codex (SEQ ID NO:25 and 26). The underlined sequences are sensestrands (bald letters) and antisense strands of dsRNAs resulting aftercleavage with a dicer (not containing the 3′-overhang “TT”). Smallletters indicate a mismatch or a loop.

[SEQ ID NO: 5] 5′-TTT GACTGGATGACTGCCATGG ttcaagagaCCATGGCAGTCATCCAGTCTTTTTT-3′ [SEQ ID NO: 6] 5'-TTT GATATCCTGCCATCACCTCttcaagagaGAGGTGATGGCAGGA TATCTTTTTT-3′ [SEQ ID NO: 7] 5'-TTTGGCCCAAGTGAAGCCCTCC ttcaagagaGGAGGGCTTCACTTG GGCCTTTTTT-3′[SEQ ID NO: 8] 5'-TTT GTGAAGCCCTCCGAGTGTC ttcaagagaGACACTCGGAGGGCTTCACTTTTTT-3′ [SEQ ID NO: 9] 5'-TTT GCCCTCCGAGTGTCAGGAGttcaagagaCTCCTGACACTCGGA GGGCTTTTTT-3′ [SEQ ID NO: 10] 5'-TTTGTCTGTTATGTGCACGTAC ttcaagagaGTACGTGCACATAAC AGACTTTTTT-3′[SEQ ID NO: 11] 5'-TTT GTACTCTCCTCCCCTCAAT ttcaagagaATTGAGGGGAGGAGAGTACTTTTTT-3′ [SEQ ID NO: 12] 5'-TTT GCTATTCTGCCAGCTGGCGttcaagagaCGCCAGCTGGCAGAA TAGCTTTTTT-3′ [SEQ ID NO: 13] 5'-TTTGACGTGCCCTGTGCAGTTG ttcaagagaCAACTGCACAGGGCA CGTCTTTTTT-3′[SEQ ID NO: 14] 5'-TTT GAAGTCACAGCACATGACG ttcaagagaCGTCATGTGCTGTGACTTCTTTTTT-3′ [SEQ ID NO: 15] 5'-TTT GTCACAGCACATGACGGAGttcaagagaCTCCGTCATGTGCTG TGACTTTTTT-3′ [SEQ ID NO: 16] 5'-TTTGGAAATTTGTATCCCGAGT ttcaagagaACTCGGGATACAAAT TTCCTTTTTT-3′[SEQ ID NO: 17] 5'-TTT GTACATGTGTAATAGCTCC ttcaagagaGGAGCTATTACACATGTACTTTTTT-3′ [SEQ ID NO: 18] 5'-TTT GACTCCAGTGGGAACCTTCttcaagagaGAAGGTTCCCACTGG AGTCTTTTTT-3′ [SEQ ID NO: 19] 5'-TTTGTCCTTTGCCCTGAACTGC ttcaagagaGCAGTTCAGGGCAAA GGACTTTTTT-3′[SEQ ID NO: 20] 5'-TTT GATCCGCGGGCGTAAACGC ttcaagagaGCGTTTACGCCCGCGGATCTTTTTT-3′ [SEQ ID NO: 21] 5'-TTT GACCAAGAAGGGCCAGTCTttcaagagaAGACTGGCCCTTCTT GGTCTTTTTT-3′ [SEQ ID NO: 22] 5'-TTTGAAAGTGGGGCCTGACTCA ttcaagagaTGAGTCAGGCCCCAC TTTCTTTTTT-3′[SEQ ID NO: 23] 5'-TTT GTTGGGGAATAGGTTGATA ttcaagagaTATCAACCTATTCCCCAACTTTTTT-3′ [SEQ ID NO: 24] 5'-TTT GATTCTATCTTGGGCCCTCttcaagagaGAGGGCCCAAGATAG AATCTTTTTT-3′ [SEQ ID NO: 25] 5'-TTTG CAuTACAgGTACgTGTGTA gtgtgctgtccTACACATGTACT TGTAGTGTTTTTT-3′[SEQ ID NO: 26] 5′-TTTG CAGTuTACTT uCCGCCgT A gtgtgctgtccTATGGCGGGAAGTAGACTGTTTTTT-3′

An siRNA against p53 can be prepared by synthesizing a sense strandoligonucleotide and antisense strand oligonucleotide designed asdescribed above using an automated DNA/RNA synthesizer, respectively,and, for example, denaturing the oligonucleotides in an appropriateannealing buffer solution at about 90 to about 95° C. for about 1minute, thereafter annealing the same at about 30 to about 70° C. forabout 1 to about 8 hours. An shRNA against p53 can be prepared bysynthesizing oligonucleotides having an shRNA sequence, designed asdescribed above, using an automated DNA/RNA synthesizer, and allowingthe same to self-anneal as described above.

Although the nucleotide molecules that constitute the siRNA and shRNAmay be naturally occurring RNAs, the molecules can comprise variouschemical modifications in order to increase the stability (chemicaland/or to-enzyme) or specific activity (affinity for mRNA). For example,to prevent degradation by hydrolylases such as nuclease, the phosphoricacid residue (phosphate) of each nucleotide that constitutes the siRNAor shRNA can be substituted with, for example, a chemically modifiedphosphoric acid residue such as phosphorothioate (PS),methylphosphonate, or phosphorodithionate. The hydroxyl group at the2′-position of the sugar (ribose) of each nucleotide may be replacedwith —H or —OR (R represents, for example, CH₃(2′-O-Me),CH₂CH₂OCH₃(2′-O-MOE), CH₂CH₂NHC(NH)NH₂, CH₂CONHCH₃, CH₂CH₂CN or thelike). Furthermore, a base moiety (pyrimidine, purine) may be chemicallymodified; for example, introduction of a methyl group or a cationicfunctional group into the 5-position of the pyrimidine base,substitution of the 2-position carbonyl group with thiocarbonyl and thelike can be mentioned.

Regarding the conformation of the sugar moiety of RNA, two types aredominant: C2′-endo (S type) and C3′-endo (N type); in a single-strandedRNA, the sugar moiety occurs in an equilibrium of both, but when adouble strand is formed, the conformation is fixed at the N type.Therefore, BNA (LNA) (Imanishi, T. et al., Chem. Commun., 1653-9, 2002;Jepsen, J. S. et al., Oligonucleotides, 14, 130-46, 2004) and ENA(Morita, K. et al., Nucleosides Nucleotides Nucleic Acids, 22, 1619-21,2003), which are RNA derivatives wherein the conformation of the sugarmoiety is fixed at the N type by bridging the 2′ oxygen and 4′ carbon soas to confer strong bindability to the target RNA, can also be usedpreferably.

However, because replacing all ribonucleoside molecules in a naturallyoccurring RNA with modified type molecules can lead to the loss of RNAiactivity, it is necessary to introduce a nucleoside modified to theminimum possible extent that allows the RISC complex to function.

An siRNA against p53 can also be purchased from, for example, Ambion(e.g., Ambion Cat #AM16708, an siRNA ID #69659, 69753, 69843, 187424,187425, 187426), Santa Cruz (e.g., Santa Cruz Cat #sc-29436, 44219) andthe like.

An siRNA and shRNA against human p53 can also be designed andsynthesized using one of the aforementioned search software programs, byinputting the sequence of human p53 cDNA shown by SEQ ID NO:3 or Refseq.No. (NM_(—)000546) and the like as a query, or can also be purchasedfrom Ambion and the like. Specifically, an shRNA against human p53having the sequence5′-GACTCCAGTGGTAATCTACTGCTCGAGCAGTAGATTACCACTGGAGTC-3′ (SEQ ID NO: 28;the bald letters indicate the target sequence for p53; underlined arethe portions where a dsRNA is formed), the shRNA against p53 describedin Science, 296, 550-553 (2002), and the like can be mentioned.

Contact of an siRNA or shRNA against p53 with a somatic cell can beachieved by, as in the case of plasmid DNA, introducing the nucleic acidinto the cell using the liposome method, polyamine method,electroporation method, beads method and the like. The method using acationic liposome is the most common and offers high transferefficiency. In addition to common transfection reagents such asLipofectamine-2000 and Oligofectamine (Invitrogen), for example,transfer reagents suitable for introduction of an siRNA, such as theGeneEraser™ siRNA transfection reagent (Stratagene), are alsocommercially available.

(b2) Vectors that Express an siRNA or shRNA Against p53

Vectors that express an siRNA are available in the tandem type and thestem loop (hairpin) type. The former is the type in which an expressioncassette for a sense strand of an siRNA and an expression cassette foran antisense strand are ligated tandem, each strand being expressed inthe cell and undergoing annealing to form a double-stranded siRNA(dsRNA). Meanwhile, the latter is the type in which an expressioncassette for an shRNA is inserted into a vector, the shRNA beingexpressed in the cell and undergoing processing by a dicer to form adsRNA. Although a polII promoter (for example, immediate-early promoterof CMV) may be used as the promoter, it is common practice to use apolIII promoter in order to allow the accurate transcription of shortRNA. As the polIII promoter, mouse and human U6-snRNA promoters, humanH1-RNase P RNA promoter, human valine-tRNA promoter and the like can bementioned. As a transcription termination signal, a sequence of 4 ormore T residues in succession is used.

The siRNA or shRNA expression cassette thus constructed is then insertedinto a plasmid vector or a viral vector. As such vectors, the same asthose described with respect to a nucleic acid that encodes a dominantnegative mutant of p53 can be utilized preferably (viral vectors such asretrovirus, lentivirus, adenovirus, adeno-associated virus, herpesvirus,and Sendai virus; animal cell expression plasmids and the like). Thevector used can be chosen as appropriate according to the intended useof the iPS cell obtained, as in the case of a dominant negative mutant.Alternatively, as an expression vector that encodes an shRNA againstp53, a viral vector such as retrovirus, prepared on the basis of acommercially available plasmid (for example, pMKO.1-puro p53 shRNA2:#10672, commercially available from Addgene, and the like) or the likecan also be used. The aforementioned Cre-loxP system or piggyBactransposon system can also be utilized as required.

Contact of a vector that expresses an siRNA or shRNA against p53 with asomatic cell is achieved by introducing a plasmid vector or viral vectorprepared as described above into the cell. Transfer of these genes canbe achieved by the same technique as that described with respect to anucleic acid that encodes a dominant negative mutant of p53.

(b3) Other Substances

As other substances that inhibit the expression of the p53 gene,antisense nucleic acids against p53 and ribozymes can be mentioned.

The antisense nucleic acid may be a DNA or an RNA, or a DNA/RNA chimera.When the antisense nucleic acid is a DNA, an RNA:DNA hybrid formed by atarget RNA and the antisense DNA is capable of being recognized byendogenous RNase H to cause selective degradation of the target RNA.Therefore, in the case of an antisense DNA to be degraded with RNase H,the target sequence may be not only a sequence in p53 mRNA, but also asequence in the intron region of the primary transcript of the p53 gene.The length of the target region for the antisense nucleic acid is notparticularly limited, as far as hybridization of the antisense nucleicacid results in an inhibition of the translation into the p53 protein;the target region may be the entire sequence or a partial sequence ofp53 mRNA, and may be a sequence of about 15 bases for the shortest, orof the entire sequence of the mRNA or primary transcript for thelongest. Considering the ease of synthesis, antigenicity,transferability in cells and other issues, an oligonucleotide consistingof about 15 to about 40 bases, particularly about 18 to about 30 bases,is preferable. Positions of the target sequence include, but are notlimited to, 5′- and 3′-UTR, vicinities of the start codon and the like.

A ribozyme refers to an RNA possessing an enzyme activity to cleave anucleic acid in the narrow sense, and is herein understood to be used asa concept encompassing a DNA, as far as the ribozyme possessessequence-specific nucleic acid cleavage activity. One of the mostversatile ribozymes is a self-splicing RNA found in infectious RNAs suchas viroid and virusoid, and the hammerhead type, the hairpin type andthe like are known. The hammerhead type exhibits enzyme activity withabout 40 bases in length, and it is possible to specifically cleave thetarget mRNA by making several bases at both ends adjoining to thehammerhead structure portion (about 10 bases in total) be a sequencecomplementary to the desired cleavage site of the mRNA.

An antisense nucleic acid or a ribozyme can be synthesized using anautomated DNA/RNA synthesizer. The nucleotide molecules that constitutethem may also have the same modifications as those for siRNA, so as toincrease the stability, specific activity and the like.

Alternatively, the antisense nucleic acid or ribozyme can also be usedin the form of a nucleic acid that encodes the same, as in the case ofsiRNA.

The aforementioned inhibitor of p53 function needs to be brought intocontact with a somatic cell in a way sufficient to inhibit the p53function in the step of somatic cell nuclear reprogramming. Here,nuclear reprogramming of the somatic cell can be achieved by bringing anuclear reprogramming substance into contact with the somatic cell.

(c) Nuclear Reprogramming Substances

In the present invention, “a nuclear reprogramming substance” refers toany substance capable of inducing an iPS cell from a somatic cell, suchas a proteinous factor or a nucleic acid that encodes the same(including forms incorporated in a vector), or a low-molecular compound.When the nuclear reprogramming substance is a proteinous factor or anucleic acid that encodes the same, the following combinations can bementioned as preferable examples (hereinafter, only the names forproteinous factors are shown).

(1) Oct3/4, Klf4, c-Myc

(2) Oct3/4, Klf4, c-Myc, Sox2 (here, Sox2 is replaceable with Sox1,Sox3, Sox15, Sox17 or Sox18. Also, Klf4 is replaceable with Klf1, Klf2or Klf5. Furthermore, c-Myc is replaceable with T58A (active mutant),N-Myc, L-Myc.)

(3) Oct3/4, Klf4, c-Myc, Sox2, Fbx15, Nanog, Eras, ECAT15-2, Tell,β-catenin (active mutant S33Y)

(4) Oct3/4, Klf4, c-Myc, Sox2, TERT, SV40 Large T

(5) Oct3/4, Klf4, c-Myc, Sox2, TERT, HPV16 E6

(6) Oct3/4, Klf4, c-Myc, Sox2, TERT, HPV16 E7

(7) Oct3/4, Klf4, c-Myc, Sox2, TERT, HPV6 E6, HPV16 E7

(8) Oct3/4, Klf4, c-Myc, Sox2, TERT, Bmi1

(for all above, see WO 2007/069666 (however, in the combination (2)above, for replacement of Sox2 with Sox18, and replacement of Klf4 withKlf1 or Klf5, see Nature Biotechnology, 26, 101-106 (2008)). Forcombinations of “Oct3/4, Klf4, c-Myc, Sox2”, see also Cell, 126, 663-676(2006), Cell, 131, 861-872 (2007) and the like. For combinations of“Oct3/4, Klf2 (or Klf5), c-Myc, Sox2”, see also Nat. Cell Biol., 11,197-203 (2009). For combinations of “Oct3/4, Klf4, c-Myc, Sox2, hTERT,SV40 Large T”, see also Nature, 451, 141-146 (2008).)(9) Oct3/4, Klf4, Sox2 (see Nature Biotechnology, 26, 101-106 (2008).)(10) Oct3/4, Sox2, Nanog, Lin28 (see Science, 318, 1917-1920 (2007))(11) Oct3/4, Sox2, Nanog, Lin28, hTERT, SV40 Large T (see Stem Cells,26, 1998-2005 (2008))(12) Oct3/4, Klf4, c-Myc, Sox2, Nanog, Lin28 (see Cell Research (2008)600-603)(13) Oct3/4, Klf4, c-Myc, Sox2, SV40 Large T (see also Stem Cells, 26,1998-2005 (2008))(14) Oct3/4, Klf4 (see Nature 454:646-650 (2008), Cell Stem Cell,2:525-528 (2008)))(15) Oct3/4, c-Myc (see Nature 454:646-650 (2008))(16) Oct3/4, Sox2 (see Nature, 451, 141-146 (2008), WO2008/118820)(17) Oct3/4, Sox2, Nanog (see WO2008/118820)(18) Oct3/4, Sox2, Lin28 (see WO2008/118820)(19) Oct3/4, Sox2, c-Myc, Esrrb (here, Esrrb is replaceable with Esrrg;see Nat. Cell Biol., 11, 197-203 (2009))(20) Oct3/4, Sox2, Esrrb (see Nat. Cell Biol., 11, 197-203 (2009))(21) Oct3/4, Klf4, L-Myc(22) Oct3/4, Nanog(23) Oct3/4(24) Oct3/4, Klf4, c-Myc, Sox2, Nanog, Lin28, SV40LT (see Science, 324:797-801 (2009))

In (1)-(24) above, in place of Oct3/4, other members of the Oct family,for example, Oct1A, Oct6 and the like, can also be used. In place ofSox2 (or Sox1, Sox3, Sox15, Sox17, Sox18), other members of the Soxfamily, for example, Sox7 and the like, can also be used. In place ofc-Myc, other members of the Myc family, for example, L-Myc and the like,can also be used. In place of Lin28, other members of the Lin family,for example, Lin28b and the like, can also be used.

A combination that does not fall in (1)-(24) above, but contains all theconstituents in any one thereof and further comprises an optionallychosen other substance, can also be included in the scope of “nuclearreprogramming substances” in the present invention. Under conditionswherein the somatic being the subject of nuclear reprogramming isendogenously expressing one or more of the constituents in any one of(1)-(24) above at a level sufficient to secure nuclear reprogramming, acombination of the remaining constituents excluding the one or moreconstituents can also be included in the scope of “nuclear reprogrammingsubstances” in the present invention.

Among these combinations, as examples of preferable nuclearreprogramming substances, at least one, preferably two or more, morepreferably 3 or more selected from Oct3/4, Sox2, Klf4, c-Myc, Nanog,Lin28 and SV40LT can be mentioned.

With the use of the iPS cells obtained for therapeutic purposes in mind,of these combinations, the combination of the 3 factors Oct3/4, Sox2 andKlf4 (that is, (9) above) is preferable. In the method of the presentinvention, with the aforementioned 3 factors only, IFS cells can beobtained at sufficiently high efficiency. That is, even with the 3factors only, IFS cells can be established at an efficiency closer tothe efficiency with 4 factors (Oct3/4, Sox2, Klf4 and c-Myc). Meanwhile,when the use of the iPS cells for therapeutic purposes is not in mind(for example, used as an investigational tool for drug discoveryscreening and the like), as well as the aforementioned 4 factors, the 5factors Oct3/4, Klf4, c-Myc, Sox2 and Lin28, or the 6 factors includingthe 5 factors plus Nanog (that is, (12) above), are preferable. In thesepreferred combinations, L-Myc can also be used in place of c-Myc.

Mouse and human cDNA sequence information on the aforementionedproteinous factors can be acquired by referring to the NCBI accessionnumbers mentioned in WO 2007/069666 (in the publication, Nanog ismentioned with the designation “ECAT4”; mouse and human cDNA sequenceinformation on Lin28, Lin28b, Esrrb, and Esrrg can be acquired byreferring to the following NCBI accession numbers, respectively); thoseskilled in the art are easily able to isolate these cDNAs.

Name of gene Mouse Human Lin28 NM_145833 NM_024674 Lin28b NM_001031772NM_001004317 Esrrb NM_011934 NM_004452 Esrrg NM_011935 NM_001438

When a proteinous factor itself is used as a nuclear reprogrammingsubstance, the factor can be prepared by inserting the cDNA obtainedinto an appropriate expression vector, introducing the vector into ahost cell, culturing the cell, and recovering the recombinant proteinousfactor from the culture obtained. Meanwhile, when a nucleic acid thatencodes a proteinous factor is used as a nuclear reprogrammingsubstance, the cDNA obtained is inserted into a viral vector or aplasmid vector to construct an expression vector as in theaforementioned case of a nucleic acid that encodes a dominant negativemutant of p53, and the vector is subjected to the step of nuclearreprogramming. The aforementioned Cre-loxP system or piggyBac transposonsystem can be utilized as required. When nucleic acids that encode 2 ormore proteinous factors are introduced into a cell as nuclearreprogramming substances, the nucleic acids may be carried by separatevectors, and a plurality of nucleic acids may be joined tandem to obtaina polycistronic vector. In the latter case, to enable efficientpolycistronic expression, it is desirable that the 2A self-cleavingpeptide of foot-and-mouth disease virus (see Science, 322, 949-953, 2008and the like), IRES sequence and the like, preferably the 2A sequence beligated between the individual nucleic acids.

When p53 function is inhibited, transgenes integrated into chromosomesvia retoroviral or lentiviral vectors tend to resistant to genesilencing. Therefore, use of a plasmid vector is advantageous forpreventing unnecessary persistent expression of exogenous nuclearreprogramming substances.

Contact of a nuclear reprogramming substance with a somatic cell can beachieved as with the aforementioned dominant negative mutant of p53 (a)when the substance is a proteinous factor; as with the aforementionednucleic acid that encodes a dominant negative mutant of p53 (b) when thesubstance is a nucleic acid that encodes the proteinous factor (a); andas with the aforementioned chemical inhibitor of p53 (c) when thesubstance is a low-molecular compound.

As described above, an inhibitor of p53 function needs to be broughtinto contact with a somatic cell in a way sufficient to inhibit the p53function in the step of somatic cell nuclear reprogramming. As far asthis requirement is met, the nuclear reprogramming substance and theinhibitor of p53 function may be brought into contact with the somaticcell simultaneously, or either one may be brought into contact inadvance. In a mode of embodiment, for example, when the nuclearreprogramming substance is a nucleic acid that encodes a proteinousfactor, and the inhibitor of p53 function is a chemical inhibitor, theformer involves a given length of time lag from the transfectiontreatment to the mass-expression of the proteinous factor, whereas thelatter is capable of rapidly inhibiting the p53 function, so that afterthe cell is cultured for a given length of time after the transfectiontreatment, the chemical inhibitor of p53 can be added to the medium. Inanother mode of embodiment, for example, when the nuclear reprogrammingsubstance and the inhibitor of p53 function are used in the form ofviral vectors or plasmid vectors, both may be simultaneously introducedinto the cell.

The number of repeats of the manipulation to introduce an adenoviral ornon-viral expression vector into a somatic cell is not particularlylimited, the transfection can be performed once or more optionallychosen times (e.g., once to 10 times, once to 5 times or the like). Whentwo or more kinds of adenoviral or non-viral expression vectors areintroduced into a somatic cell, it is preferable that these all kinds ofadenoviral or non-viral expression vectors be concurrently introducedinto a somatic cell; however, even in this case, the transfection can beperformed once or more optionally chosen times (e.g., once to 10 times,once to 5 times or the like), preferably the transfection can berepeatedly performed twice or more (e.g., 3 times or 4 times).

(d) iPS Cell Establishment Efficiency Improvers

By bringing, in addition to an inhibitor of p53 function, anotherpublicly known iPS cell establishment efficiency improver, into contactwith a somatic cell, the efficiency of establishment of iPS cells isexpected to be increased more. Examples of iPS cell establishmentefficiency improvers include, but are not limited to, histonedeacetylase (HDAC) inhibitors [e.g., valproic acid (VPA) (Nat.Biotechnol., 26(7): 795-797 (2008)), low-molecular inhibitors such astrichostatin A, sodium butyrate, MC 1293, and M344, nucleic acid-basedexpression inhibitors such as siRNAs and shRNAs against HDAC (e.g.,HDAC1 siRNA Smartpool® (Millipore), HuSH 29mer shRNA Constructs againstHDAC1 (OriGene) and the like), and the like], G9a histonemethyltransferase inhibitors [for example, low-molecular inhibitors suchas BIX-01294 (Cell Stem Cell, 2: 525-528 (2008)), nucleic acid-basedexpression inhibitors such as siRNAs and shRNAs against G9a (e.g., G9asiRNA (human) (Santa Cruz Biotechnology) and the like) and the like],L-channel calcium agonists (for example, Bayk8644) (Cell Stem Cell, 3,568-574 (2008)), UTF1 (Cell Stem Cell, 3, 475-479 (2008)), Wnt Signaling(for example, soluble Wnt3a) (Cell Stem Cell, 3, 132-135 (2008)), 21/LIF(21 is an inhibitor of mitogen-activated protein kinase signaling andglycogen synthase kinase-3, PloS Biology, 6(10), 2237-2247 (2008)) andthe like, and the like. As mentioned above, the nucleic acid-basedexpression inhibitors may be in the form of expression vectors harboringa DNA that encodes an siRNA or shRNA.

Of the aforementioned constituents of nuclear reprogramming substances,SV40 large T, for example, can also be included in the scope of iPS cellestablishment efficiency improvers because they are auxiliary factorsunessential for the nuclear reprogramming of somatic cells. While themechanism of nuclear reprogramming remains unclear, it does not matterwhether auxiliary factors, other than the factors essential for nuclearreprogramming, are deemed nuclear reprogramming substances, or deemediPS cell establishment efficiency improvers. Hence, because the somaticcell nuclear reprogramming process is visualized as an overall eventresulting from contact of nuclear reprogramming substances and an iPScell establishment efficiency improver with somatic cells, it does notalways seem necessary for those skilled in the art to distinguish both.

Contact of these other iPS cell establishment efficiency improvers witha somatic cell can be achieved as described above with respect tofunctional inhibitors of p53, respectively, when the improver is (a) aproteinous factor, (b) a nucleic acid that encodes the proteinousfactor, or (c) a low-molecular compound.

(e) Source of Somatic Cells

The somatic cells that can be used as a starting material for thepreparation of iPS cells in the present invention may be any cells,other than germ cells, derived from a mammal (for example, mouse orhuman); for example, keratinizing epithelial cells (e.g., keratinizedepidermal cells), mucosal epithelial cells (e.g., epithelial cells ofthe superficial layer of tongue), exocrine gland epithelial cells (e.g.,mammary gland cells), hormone-secreting cells (e.g., adrenomedullarycells), cells for metabolism or storage (e.g., liver cells), intimalepithelial cells constituting interfaces (e.g., type I alveolar cells),intimal epithelial cells of the obturator canal (e.g., vascularendothelial cells), cells having cilia with transporting capability(e.g., airway epithelial cells), cells for extracellular matrixsecretion (e.g., fibroblasts), constrictive cells (e.g., smooth musclecells), cells of the blood and the immune system (e.g., T lymphocytes),sense-related cells (e.g., bacillary cells), autonomic nervous systemneurons (e.g., cholinergic neurons), sustentacular cells of sensoryorgans and peripheral neurons (e.g., satellite cells), nerve cells andglia cells of the central nervous system (e.g., astroglia cells),pigment cells (e.g., retinal pigment epithelial cells), and progenitorcells thereof (tissue progenitor cells) and the like can be mentioned.There is no limitation on the degree of cell differentiation; evenundifferentiated progenitor cells (including somatic stem cells) andfinally differentiated mature cells can be used alike as a source ofsomatic cells in the present invention. Here, as examples ofundifferentiated progenitor cells, tissue stem cells (somatic stemcells) such as nerve stem cells, hematopoietic stem cells, mesenchymalstem cells, and dental pulp stem cells can be mentioned.

According to the method of the present invention, iPS cells can beefficiently obtained even from finally differentiated somatic cells, forwhich iPS cells are reportedly generally difficult to establish. In apreferred mode of embodiment of the present invention, a T cell is usedas a somatic cell. The T cell may be CD4-positive or CD8-positive, andmay be a cell in a CD4/CD8 double-positive differentiation stage. Tcells can be isolated from the spleen, lymph node, peripheral blood,cord blood and the like by a method known per se, for example flowcytometry using an antibody against a cell surface marker such as CD4,CD8, or CD3, and a cell sorter. In the case of mice, it is preferablethat a somatic cell be collected from the spleen or lymph node, in whichthe content ratio of T cells is high; however, in the case of humans, itis desirable, from the viewpoint of the low invasiveness and the ease ofpreparation, that a T cell be prepared from peripheral blood, cord bloodor the like.

The choice of mammal serving as a source of somatic cells collected isnot particularly limited; however, when the iPS cells obtained are usedfor regenerative medicine in humans, it is particularly preferable, fromthe viewpoint of non-occurrence of graft rejection, that a somatic cellbe collected from a patient or another person of the same HLA type. Whenthe IPS cells are not administered (transplanted) to a human, but usedas, for example, a source of cells for screening for evaluating apatient's drug susceptibility or the presence or absence of an adversereaction, it is likewise necessary to collect a somatic cell from apatient or another person with the same genetic polymorphism correlatingto the drug susceptibility or adverse reaction.

Somatic cells separated from a mouse or a human can be pre-culturedusing a medium known per se suitable for the cultivation thereofdepending on the kind of the cells. Examples of such media include, butare not limited to, a minimal essential medium (MEM) containing about 5to 20% fetal calf serum, Dulbecco's modified Eagle medium (DMEM),RPMI1640 medium, 199 medium, F12 medium and the like. Reports areavailable that by conducting pre-culture at a low serum concentration of5% or less, the efficiency of establishment of iPS cells was improved(for example, WO 2009/006997). When a T cell is used as the somaticcell, it is desirable that the cell be pre-cultured using a mediumcontaining a cytokine such as interleukin (IL)-2, IL-7, stem cell factor(SCF), or Flt3 ligand. When using, for example, a transfection reagentsuch as a cationic liposome in the contact with a nuclear reprogrammingsubstance and an inhibitor of p53 function (and another substance thatimproves the efficiency of establishment of iPS cells), it is sometimespreferable that the medium be previously replaced with a serum-freemedium to prevent a reduction in the transfer efficiency. After thenuclear reprogramming substance is brought into contact, the cell can becultured under conditions suitable for cultivation of, for example, EScells. In the case of mouse cells, it is preferable that the culture becarried out with the addition of Leukemia Inhibitory Factor (LIF) as adifferentiation suppression factor to an ordinary medium. Meanwhile, inthe case of human cells, it is desirable that basic fibroblast growthfactor (bFGF) and/or stem cell factor (SCF) be added in place of LIF.Usually, the cell is cultured in the co-presence of fetal-mouse-derivedfibroblasts (MEF) treated with radiation or an antibiotic to terminatethe cell division, as feeder cells. As the MEF, usually STO cells andthe like are commonly used, but for inducing iPS cells, SNL cells(McMahon, A. P. & Bradley, A. Cell 62, 1073-1085 (1990)) and the likeare commonly used. Co-culture with feeder cells may be started beforecontact of the nuclear reprogramming substance, at the time of thecontact, or after the contact (for example, 1-10 days later).

A candidate colony of iPS cells can be selected by a method with drugresistance and reporter activity as indicators, and also by a methodbased on macroscopic examination of morphology. As an example of theformer, a colony positive for drug resistance and/or reporter activityis selected using a recombinant cell wherein a drug resistance geneand/or a reporter gene is targeted to the locus of a gene highlyexpressed specifically in pluripotent cells (for example, Fbx15, Nanog,Oct3/4 and the like, preferably Nanog or Oct3/4). As examples of suchrecombinant cells, a mouse-derived MEF wherein the βgeo (which encodes afusion protein of β-galactosidase and neomycin phosphotransferase) geneis knocked-in to the Fbx15 gene locus (Takahashi & Yamanaka, Cell, 126,663-676 (2006)), or a transgenic mouse-derived MEF wherein green thefluorescent protein (GFP) gene and the puromycin resistance gene areintegrated in the Nanog gene locus (Okita et al., Nature, 448, 313-317(2007)) and the like can be mentioned. Meanwhile, methods of macroscopicexamination of morphology include, for example, the method described byTakahashi et al. in Cell, 131, 861-872 (2007). Although methods usingreporter cells are convenient and efficient, colony selection bymacroscopic examination is desirable from the viewpoint of safety wheniPS cells are prepared for the purpose of human treatment. When the 3factors Oct3/4, Klf4 and Sox2 are used as nuclear reprogrammingsubstances, the number of clones established sometimes decreases, butthe resulting colonies are for the most part iPS cells whose quality isas high as that of ES cells; therefore, it is possible to efficientlyestablish iPS cells even without using reporter cells.

The identity of the cells of the selected colony as iPS cells can beconfirmed by positive responses to Nanog (or Oct3/4, Fbx15) reporters(GFP positivity, β-galactosidase positivity and the like) and positiveresponses to selection markers (puromycin resistance, G418 resistanceand the like), as well as by the formation of a visible ES cell-likecolony, as described above; however, to increase the accuracy, it ispossible to perform tests such as analyzing the expression of variousES-cell-specific genes, and transplanting the cells selected to a mouseand confirming teratoma formation. These test methods are obvious tothose skilled in the art, representative confirmatory tests beingdescribed in Examples below.

The iPS cells thus established can be used for various purposes. Forexample, by utilizing a method of differentiation induction reportedwith respect to ES cells, differentiation into various cells (e.g.,myocardial cells, blood cells, nerve cells, vascular endothelial cells,insulin-secreting cells and the like) from iPS cells can be induced.Therefore, inducing iPS cells using a somatic cell collected from apatient or another person of the same HLA type would enable stem celltherapy by autogeneic or allogeneic transplantation, wherein the iPScells are differentiated into desired cells (that is, cells of anaffected organ of the patient, cells that have a therapeutic effect ondisease, and the like), which are transplanted to the patient.Furthermore, because functional cells (e.g., hepatocytes) differentiatedfrom iPS cells are thought to better reflect the actual state of thefunctional cells in vivo than do corresponding existing cell lines, theycan also be suitably used for in vitro screening for the effectivenessand toxicity of pharmaceutical candidate compounds and the like.

When inhibition of p53 is achieved in a mode wherein a dominant negativemutant of p53 or a nucleic acid that encodes an shRNA or shRNA againstp53 or the like is introduced into a somatic cell and forcibly expressedtherein, the iPS cell obtained is a novel cell distinct fromconventionally known iPS cells because of the containment of theexogenous nucleic acid. In particular, when the exogenous nucleic acidis introduced into the somatic cell using a retrovirus, lentivirus orthe like, the exogenous nucleic acid is usually integrated in the genomeof the iPS cell obtained, so that the phenotype of containing theexogenous nucleic acid is stably retained. When the exogenous nucleicacid is introduced into the somatic cell using a persistent Sendai viralvector, the exogenous nucleic acid can occur stably in the cytoplasm ofthe iPS cell obtained, so that the phenotype of containing the exogenousnucleic acid is likewise stably retained.

The present invention also provides an iPS cell wherein the TCR gene hasbeen rearranged, the cell obtained by reprogramming a T cell. As amethod of T cell reprogramming, a method wherein a T cell is broughtinto contact with a nuclear reprogramming substance under conditions forinhibiting the p53 function as described above can be mentioned. In theiPS cell induced from a T cell (T-iPS cell), the TCR rearrangement inthe T cell from which it is derived is conserved. Although there havebeen some cases where iPS cells were induced from B cells so far, noreport has been presented regarding the establishment of iPS cellsderived from a T cell. Because the rearrangement of TCR is conservedeven in the cells differentiated from a T-iPS cell, T cells capable ofspecifically injuring cells that present one of the above-describedpeptides (e.g., cancer cells, infected cells and the like) can beproduced in large amounts by, for example, establishing a T-iPS cellfrom a T cell clone capable of specifically recognizingantigen-presenting cells (e.g., macrophages, dendritic cells and thelike) pulsated with a cancer antigen peptide or with a peptide derivedfrom a cell surface antigen of a pathogen such as a virus, amplifyingthe iPS cell to large amounts in vitro using the method of the presentinvention, then inducing their differentiation into T cells, and can beprepared as a T cell immunotherapeutic agent as with conventional T cellimmunotherapy.

The present invention is explained in more detail in the following byreferring to Examples, which are not to be construed as limitative.

EXAMPLES Example 1 Effects of p53 Deficiency

A p53 homo-deficient mouse or hetero-deficient mouse having a Nanogreporter was used as an experimental system. p53 is a gene that isexpressed in almost all cells and controls cell cycle termination orapoptosis induction during repair of damaged cells. These mice have beendeprived of the function of the p53 gene by replacing the exon 5 withthe neomycin resistance gene (Lawrence A. Donehower (1992). Nature 356,215-221). It has been reported that p53 homo-deficient mice are bornnormally but frequently suffer from tumorigenesis. The Nanog reporterwas prepared by inserting green fluorescent protein (EGFP) and thepuromycin resistance gene into the Nanog locus of a BAC (bacterialartificial chromosome) purchased from BACPAC Resources (Okita K. et al.,Nature 448, 313-317 (2007)). The mouse Nanog gene is a gene expressedspecifically in pluripotent cells such as ES cells and early embryos.The mouse iPS cells that have become positive for this reporter areknown to have a potential for differentiation nearly equivalent to thatof ES cells. By preparing a Nanog reporter mouse having this Nanogreporter (Okita K. et al., Nature 448, 313-317 (2007)), and mating thismouse with p53 deficient mice, p53 homo-deficient mice andhetero-deficient mice having the Nanog reporter were prepared.

The retroviruses used for reprogramming were prepared by introducingrespective retrovirus expression vectors (pMXs-Oct3/4, pMXs-Sox2,pMXs-Klf4, pMXs-cMyc: Cell, 126, 663-676 (2006)) into Plat-E cells(Morita, S. et al., Gene Ther. 7, 1063-1066) that had been sown to6-well culture plates (Falcon) at 0.6×10⁶ cells per well on the daybefore. The culture broth used was DMEM/10% FCS (DMEM (Nacalai tesque)supplemented with 10% fetal bovine serum), and the cells were culturedat 37° C. and 5% CO₂. For vector transfer, 4.5 μL of the FuGene6transfection reagent (Roche) was placed in 100 μL of Opti-MEM IReduced-Serum Medium (Invitrogen), and this mixture was allowed to standat room temperature for 5 minutes. Thereafter, 1.5 μg of each expressionvector was added, and the mixture was further allowed to stand at roomtemperature for 15 minutes, and then added to the Plat-E culture broth.On day 2, the Plat-E supernatant was replaced with a fresh supply of themedium; on day 3, the culture supernatant was recovered and filteredthrough a 0.45 μm sterile filter (Whatman), polybrene (Nacalai) wasadded to obtain a concentration of 4 μg/mL, and this was used as theviral liquid.

Fibroblasts (MEFs) were isolated from a fetal p53 homo-deficient mousehaving the mouse Nanog reporter (13.5 days after fertilization). Becauseof the absence of expression of the Nanog gene, MEFs do not express EGFPand do not emit green fluorescence. Because of the absence of expressionof the puromycin resistance gene as well, MEFs are susceptible topuromycin, an antibiotic. As such, the MEFs were sown to a 6-wellculture plate (Falcon) coated with 0.1% gelatin (Sigma) at 1×10⁵ perwell. The culture broth used was DMEM/10% FCS, and the cells werecultured at 37° C. and 5% CO₂. The following day, the retrovirus liquidwas added to cause overnight infection to introduce the gene.

Starting on day 3 after the viral infection, the cells were culturedusing an LIF-supplemented ES cell culture medium (one prepared by addingto DMEM (Nacarai tesque) 15% fetal bovine serum, 2 mM L-glutamine(Invitrogen), 100 μM non-essential amino acids (Invitrogen), 100 μM2-mercaptoethanol (Invitrogen), 50 U/mL penicillin (Invitrogen) and 50μg/mL streptomycin (Invitrogen)). On day 5 after the infection, themedium for the MEFs was removed, and the cells were washed by theaddition of 1 mL of PBS. After the PBS was removed, 0.25% Trypsin/1 mMEDTA (Invitrogen) was added, and a reaction was allowed to proceed at37° C. for about 5 minutes. After the cells floated up, the cells weresuspended by the addition of ES cell culture medium, and 5×10³ cellswere sown to a 100-mm dish with feeder cells sown thereto previously.The feeder cells used were SNL cells that had been treated withmitomycin C to terminate the cell division (McMahon, A. P. & Bradley, A.Cell 62, 1073-1085 (1990)). Subsequently, the ES cell culture medium wasexchanged with a fresh supply every two days until a colony wasobservable. Selection with puromycin (1.5 μg/mL) was performed, startingon day 13 for the cells infected with 4 factors (Oct3/4, Sox2, Klf4,c-Myc), and on day 19 for the cells infected with 3 factors (Oct3/4,Sox2, Klf4). Colonies were visible about on day 10 for the 4 factors,and about on day 20 for the 3 factors, and became GFP-positivegradually.

GFP-positive colonies were counted on day 21 for the 4 factors, and onday 28 for the 3 factors. The total results of three experiments areshown in FIG. 1. Compared with the p53 hetero-deficient cellsconstituting the control group, in the p53 homo-deficient cells,GFP-positive colonies increased about 2 times for the 4 factors, andabout 9 times for the 3 factors.

Next, GFP-positive colonies were collected on day 21 for the 4 factors,and on day 28 for the 3 factors, and, after trypsinization, thecultivation was continued on the feeder cells (this time point was takenas subculture generation 1). When 1×10⁶ cells were subcutaneouslyinjected to immunodeficient mice, teratoma formation began to beobservable 2 weeks later; whether the 3 factors or the 4 factors wereintroduced, the resulting GFP-positive colonies were identified as iPScells. The results for the 4 factors are shown in FIG. 2.

From the results above, it was demonstrated that by deleting(knock-outing) p53, the efficiency of establishment of iPS cells wasincreased.

Example 2 Investigation with a Dominant Negative Mutant

The function of endogenous p53 was inhibited using a dominant negativemutant of p53, and its influence on the efficiency of establishment ofiPS cells was investigated. The dominant negative mutant used wasp53P275S, prepared by causing a point mutation of the proline at theposition 275 located in the genome-binding region of p53 to serine(Annemieke de Vries (2002). PNAS 99, 2948-2953).

The retrovirus used for reprogramming was prepared by introducingretrovirus expression vector (pMXs-Oct3/4, pMXs-Sox2, pMXs-Klf4,pMXs-cMyc, pMXs-p53P275S) into Plat-E cells that had been sown to a6-well culture plate (Falcon) at 0.6×10⁶ cells per well on the previousday (for how to prepare pMXs-p53P275S, see Example 6 below). The culturebroth used was DMEM/10% FCS (DMEM (Nacalai tesque) supplemented with 10%fetal bovine serum), and cultured at 37° C. and 5% CO₂. For vectortransfer, 4.5 μL of the FuGene6 transfection reagent (Roche) was placedin 100 μl, of Opti-MEM I Reduced-Serum Medium (Invitrogen), and thismixture was allowed to stand at room temperature for 5 minutes.Thereafter, 1.5 μg of each expression vector was added, and the mixturewas further allowed to stand at room temperature for 15 minutes, andthen added to Plat-E culture broth. On day 2, the Plat-E supernatant wasexchanged with a fresh supply of the medium; on day 3, the culturesupernatant was recovered and filtered through a 0.45 μm sterile filter(Whatman), polybrene (Nacalai) was added to obtain a concentration of 4μg/mL, and this was used as the viral liquid.

Fibroblasts (MEFs) were isolated from a fetal p53 hetero-deficient mousehaving a Nanog reporter (13.5 days after fertilization). A 6-wellculture plate with feeder cells sown thereto previously was provided,and MEFs were sown at 4×10³ cells per well for the 4 factors, and at2×10⁴ cells per well for the 3 factors. The feeder cells used were SNLcells that had been treated with mitomycin C to terminate the celldivision. The culture broth used was DMEM/10% FCS, and were cultured at37° C. and 5% CO₂. The following day, the cells were cultured in a viralliquid recovered from Plat-E overnight to introduce the gene. Startingon day 3 after the infection, the cells were cultured using anLIF-supplemented ES cell culture medium. Subsequently, the ES cellculture medium was exchanged with a fresh supply every two days until acolony was visible. Selection with puromycin (1.5 μg/mL) was performedstarting on day 13 after the infection for the cells infected with both4 factors (Oct3/4, Sox2, Klf4, c-Myc) and P275S, and on day 19 for thecells infected with both 3 factors (Oct3/4, Sox2, Klf4) and P275S.Colonies were visible about on day 10 for the 4 factors, and about onday 20 for the 3 factors, and became GFP-positive gradually.

GFP-positive colonies were counted on day 21 for the 4 factors, and onday 28 for the 3 factors. An outline of the experimental procedure isshown in FIG. 3(A), and experimental results are shown in FIGS. 3(B) to(D). Compared with the cells incorporating mutation-free p53 or redfluorescent protein (DsRedExpress) in place of p53P275S, in the cellsincorporating P275S, the number of GFP-positive colonies increased about4 times for the 4 factors, and about 3 times for the 3 factors.

The results for introduction of both the above-described 3 factors or 4factors and p53 or DsRedExpress into the MEFs isolated from a fetal p53homo-deficient mouse are shown in FIGS. 3(E) to (G). Compared with thecells incorporating the control DsRedExpress, in the cells incorporatingp53, the number of GFP-positive colonies decreased.

From the results above, it was demonstrated that by inhibiting thefunction of endogenous p53, the efficiency of establishment of iPS cellswas increased.

Example 3 Investigation Using p53 Inhibitory Agent

The effects of a p53 inhibitor on the efficiency of establishment of iPScells were examined. The retrovirus used for reprogramming was preparedby introducing a retrovirus expression vector (pMXs-Oct3/4, pMXs-Sox2,pMXs-Klf4, pMXs-cMyc) into Plat-E cells that had been sown to a 6-wellculture plate (Falcon) at 0.6×10⁶ cells per well on the previous day.The culture broth used was DMEM/10% FCS (DMEM (Nacalai tesque)supplemented with 10% fetal bovine serum), and cultured at 37° C. and 5%CO₂. For vector transfer, 4.5 μL of the FuGene6 transfection reagent(Roche) was placed in 100 μL of Opti-MEM I Reduced-Serum Medium(Invitrogen), and this mixture was allowed to stand at room temperaturefor 5 minutes. Thereafter, 1.5 μg of each expression vector was added,and the mixture was further allowed to stand at room temperature for 15minutes, and then added to Plat-E culture broth. On day 2, the Plat-Esupernatant was exchanged with a fresh supply of the medium; on day 3,the culture supernatant was recovered and filtered through a 0.45 μmsterile filter (Whatman), polybrene (Nacalai) was added to obtain aconcentration of 4 μg/ml, and this was used as the viral liquid.

Fibroblasts (MEF) were isolated from a fetal p53 hetero-deficient mousehaving the Nanog reporter (13.5 days after fertilization). These MEFswere sown to a 6-well culture plate (Falcon) coated with 0.1% gelatin(Sigma) at 1×10⁵ cells per well. The culture broth used was DMEM/10%FCS, and the cells were cultured at 37° C. and 5% CO₂. The followingday, the cells were infected with the viral liquid recovered from Plat-Eovernight to introduce the gene. Starting on day 3 after the infection,the cells were cultured using an LIF-supplemented ES cell culturemedium. On day 5 after the infection, the medium for MEF was removed,and the cells were washed by the addition of 1 mL of PBS. After the PBSwas removed, 0.25% Trypsin/1 mM EDTA (Invitrogen) was added, and areaction was allowed to proceed at 37° C. for about 5 minutes. After thecells floated up, the cells were suspended by the addition of ES cellculture medium, and 8×10² cells were sown to a 6-well culture plate withfeeder cells sown thereto previously. Starting on day 6, Pifithrin α,p-Nitro, Cyclic (MERCK), 6 nM, was added to the medium, and thecultivation was continued. Pifithrin was used in solution in DMSO.Starting on day 16 after the infection, selection with puromycin (1.5μg/mL) was performed. Colonies were visible on day 10, and becameGFP-positive gradually. GFP-positive colonies were counted on day 20.The results are shown in FIG. 4. Compared with the cells treated withDMSO, in the cells treated with Pifithrin, the number of GFP-positivecolonies increased about 4 times. From the results above, it wasdemonstrated that by inhibiting the p53 function, the efficiency ofestablishment of iPS cells was increased.

Example 4 Effects of p53 Deficiency in Establishment of iPS from T Cell

A Nanog reporter mouse (hereinafter, Nanog-GFP Tg mouse: 17-week-oldmale) and a p53 homo-deficient mouse having a Nanog reporter(hereinafter, Nanog-GFP/Trp53^(−/−) mouse: 24-week-old female) wereeuthanized, after which the spleens were extirpated. After the tissuewas mechanically ground, CD90-positive cells were obtained using CD90microbeads (Miltenyi biotec) and the MACS system. 1×10⁶ cells weresuspended in T cell culture medium (DMEM, 10% FBS, 10 μl/1×10⁶ cellsCD3/CD28 T cell expander (Invitrogen), 10 units/ml IL-2) and sown to a24-well plate coated with retronectin (50 μg/ml, Takara) (1×10⁶cells/well).

The following day, the medium was replaced with 1 ml of a mediumcontaining a retrovirus harboring 4 factors (Oct3/4, Sox2, Klf4, c-Myc)(prepared in the same manner as Example 1, virus-containing supernatantsupplemented with 8 μg/ml polybrene and 10 units/ml IL-2),centrifugation was performed at 3000 rpm for 30 minutes (spin infectionmethod), and the cells were cultured at 37° C. overnight. The followingday, the medium was exchanged with a T cell culture medium, andthereafter medium exchange was performed every two days.

On day 12 after the establishment of T cells, the cells were re-sownonto mitomycin-treated SNL-PH cells (5×10⁴ cells/100-mm dish). Startingon the following day, the cells were cultured with an ES mediumsupplemented with 1.5 μg/ml puromycin.

On day 17 after the establishment of T cells, 11 GFP-positive colonieswere observed, which were picked up and sown to a 24-well SNL-PH plate.As a result, 3 clones of ES-like cells were obtained (408E2, E7, E8).These clones exhibited a mouse ES cell-like morphology and were positivefor Nanog-GFP (FIG. 5). The expression of ES cell-specific genes(Oct3/4, Sox2, Nanog, Cripto, Dax1, ERas, Fgf4, Esg1, Rex1) wasconfirmed by RT-PCR (FIG. 6). The silencing of the exogenous genesincorporated was incomplete (FIG. 6). None of these clones expressed a Tcell marker (FasL, GzmA, GzmB, Ifng) (Rever Tra Ace kit, Takara, wasused). Staining with anti-SSEA1 antibody (Santacruz) confirmed theexpression thereof, and the clones were also positive for alkalinephosphatase activity (FIG. 7). Furthermore, as a result of in vitrodifferentiation induction by embryoid formation, staining using AFP (R&Dsystems), GATA4 (Santacruz), a-SMA (DAKO), Desmin (Neomarker),bIII-tubulin (Chemicon), and GFAP (DAKO) antibodies confirmed theexpression thereof; it was found that the clones possessed the potentialfor differentiating into three germ layers (FIG. 8). It was alsoconfirmed that the clones also contributed to the genesis of chimericmice (data not shown). Thus, the resulting GFP-positive colonies wereidentified as iPS cells.

When the same experiment was performed, but using Nanog-GFP Tgmouse-derived T cells, absolutely no GFP-positive colony was produced.From the experimental results above, it was demonstrated that bydeleting (inactivating) p53, the establishment of iPS cells waspromoted, and that by inactivating p53, iPS cells could be establishedfrom finally differentiated T cells.

By microinjecting iPS cells established from T cells as described aboveinto ICR-mouse-derived blastocysts, adult chimeric mice created (FIG.10). However, all chimeric mice experienced tumorigenesis within 7weeks.

Next, these established iPS cells, various tissues of the chimera, andtumors of the chimera were examined by PCR for rearrangement of the Tcell receptor gene (V-(D)-J DNA of Tcrβ gene). Specifically, thisexamination was performed in accordance with the method described inCurr Biol 11(19), 1553 (2001). Briefly, an attempt was made to detectDβ2-Jβ2 rearrangement of the Tcrβ gene by conducting PCR amplificationon genomic DNA using primer sets Dβ2 (GTAGGCACCTGTGGGGAAGAAACT; SEQ IDNO:29) and Jβ2 (TGAGAGCTGTCTCCTACTATCGATT; SEQ ID NO:30), andelectrophoresing the resulting PCR product on 1.2% agarose gel. As aresult, a band of the rearranged T cell receptor was detected; the iPScells were identified as being derived from T cells. The rearranged bandin chimera tumor was as dense as that of the iPS cells, it was suggestedthat the tumor might be derived from the iPS cells (FIG. 11).

Example 5 Microarray Analysis

To determine whether there was a difference in expression patternbetween MEFs isolated from a p53 homo-deficient mouse and MEFs isolatedfrom an ordinary, non-p53-deficient mouse, DNA microarray analysis wasperformed. The analysis was performed using total RNA derived from MEFsisolated from each mouse, by the technique described in Cell, 131,861-872 (2007). The results are shown in FIG. 9. An attempt was made todetect genes expressed specifically in ES cells (genes expressed atlevels 10 times or more higher in the ES cells than in the fibroblasts);in the MEFs derived from the p53-deficient mouse, compared with thewild-type MEFs, these genes expressed specifically in the ES cells wereexpressed much more (FIG. 9(B)). Conversely, an attempt was made todetect genes expressed specifically in fibroblasts (a group of genesexpressed at levels 10 times or more higher in the fibroblasts than inthe ES cells); in the MEFs derived from the p53-deficient mouse,compared with the wild-type MEFs, the expression of these genesexpressed specifically in the fibroblasts was extremely lower (FIG.9(C)). From the results above, it was shown that by deleting p53, astate close to ES cells was generated.

Example 6 Effects of p53 Deficiency and Introduction of a DominantNegative Mutant

We used either the Nanog-GFP or Oct3/4-GFP reporter system for sensitiveand specific identification of iPS cells (Okita K. et al. Nature 448,313 (2007)). Wild type, p53^(+/−), or p53^(−/−) MEFs, which also containthe Nanog-GFP reporter, were seeded at 1×10⁵ cells per well of 6 wellplates. The cells were infected next day (day 0) with retrovirus madefrom Plat-E. On day 5, cells were reseeded either at one cell per wellof 96-plates by a cell sorter (FACS Aria, Beckton Dekinson) or 5000cells per 100 mm-dish. Puromycin selection (1.5 μg/ml) was initiated onday 13 in the four-factor protocol and on day 19 in the three factorprotocol. Numbers of GFP positive colonies were determined on day 21 forthe four-factor protocol and on day 28 in the three factor protocol. Theresults are shown in FIG. 12.

When the three factors devoid of Myc were introduced into Nanog-GFP,p53-wild type MEF, we obtained 1˜18 GFP-positive colonies from 5000transduced fibroblasts (FIG. 12 a). From Nanog-GFP, p53-heterozygousmutant MEFs, we observed 7˜81 GFP-positive colonies. In contrast, fromNanog-GFP, p53-null fibroblasts, 38˜478 GFP-positive colonies emerged.

We next tried to generate iPS cells from a single fibroblast. By using aflow cytometer, we plated one Nanog-GFP cell (p53 wild-type,heterozygous mutant, or homozygous mutant), which was transduced withthe three factors five days before the re-plating, into a well of96-well plates. Twenty-three days after the re-plating, we observedGFP-positive colonies in zero or one well per a 96-well plate with p53wild-type fibroblasts (FIG. 12 b). By great contrast, we observedGFP-positive colonies in ˜2 and ˜10 wells per a 96-well plate withp53-heterozyous fibroblasts and p53-null fibroblasts, respectively.These data showed that loss of p53 function markedly increase theefficiency of direct reprogramming and up to 10% of transduced cells canbecome iPS cells with the three factors devoid of Myc.

We performed the same experiment with the four factors including c-Myc.We observed GFP-positive colonies in zero or one well per a 96-wellplate with p53 wild-type fibroblasts (FIG. 12 c). By great contrast, weobserved GFP-positive colonies in ˜9 and ˜25 wells per a 96-well platewith p53-heterozyous fibroblasts and p53-null fibroblasts, respectively.These data showed that addition of the Myc retrovirus further increasedthe efficiency of direct reprogramming up to 20%.

To confirm the suppressive role of p53 in iPS cell generation, weperformed two sets of experiments. First, we tested the effect ofdominant negative mutants of p53 on the generation of iPS cells.Complimentary DNA of mouse p53 gene was amplified by RT-PCR with p53-1S(CAC CAG GAT GAC TGC CAT GGA GGA GTC; SEQ ID NO:31) and p53-1223AS (gtgtct cag ccc tga agt cat aa; SEQ ID NO:32), and subcloned intopENTER-D-TOPO (Invitrogen). After sequencing verification, cDNA wastransferred to pMXs-gw by Gateway cloning technology (Invitrogen).Retroviral vectors for p53 mutants were generated by two step PCR. Forthe P275S mutant (Annemieke de Vries (2002), supra), the first PCR wasperformed with two primer sets, p53-P275S—S (tgt ttg tgc ctg ctc tgg gagaga ccg c; SEQ ID NO:33) and p53-1223AS, and p53-P275S-AS (gcg gtc tctccc aga gca ggc aca aac a; SEQ ID NO:34) and p53-1S. For the D278Nmutant (Shinmura K. et al. Oncogene 26(20), 2939 (2007)), the first PCRwas performed with two primer sets, p53-D278N-S (tgc cct ggg aga aac cgccgt aca gaa; SEQ ID NO:35) and p53-1223AS, and p53-D278N-AS (ttc tgt acggcg gtt tct ccc agg gca; SEQ ID NO:36) and p53-1S. For the S58A mutant(Cecchinelli B. et al. Cell Death Differ 13(11), 1994 (2006)), the firstPCR was performed with two primer sets, p53-S58A-S (ttt gaa ggc cca GCtgaa gcc ctc cga; SEQ ID NO:37) and p53-1223AS, and p53-S58A-AS (tcg gagggc ttc aGC tgg gcc ttc aaa; SEQ ID NO:38) and p53-1S. The two PCRproducts of each first PCR were mixed and used as a template for thesecondary PCR with the primer set, p53-1S and p53-1223AS. These mutantswere cloned into pENTR-D-TOPO, and then transferred to pMXs-gw byGateway cloning technology. The retroviruses expressing p53 mutantsobtained were co-transduced with the three factors into Oct4-GFP,p53^(+/−) or p53^(−/−) MEFs. The results are shown in FIG. 13.

When the dominant negative mutant P275S was introduced into Oct4-GFP,p53-heterozygous MEF, we observed substantial increase in the number ofGFP-positive colonies (FIG. 13 a). By contrast, the wild-type p53decreased the efficiency of iPS cell generation.

Second, we placed cDNA encoding the wild-type p53 ortransactivation-negative mutant (D278N or S58A) into the pMXs retroviralvector (Morita S. et al. Gene Ther 7(12), 1063 (2000)) and introduced ittogether with the retroviruses for the three reprogramming factors intoNanog-GFP, p53-null MEFs. We found that the wild-type p53 markedlydecreased the number of GFP-positive colonies (FIG. 13 b). Thetransactivation-negative p53 mutants, in contrast, showed weaker effectsthan did the wild-type protein. These data confirmed that loss of p53 isresponsible to the observed increase in the efficiency of directreprogramming.

Next, we randomly expanded ten clones of GFP-positive, p53-null cells,generated by the three factors. All the clones showed morphology similarto that of mouse ES cells (FIG. 14, left). The iPS cells generated bythe three factors expressed ES cell marker genes at comparable levels tothose in ES cells (FIG. 15, left). The expression of the threetransgenes was effectively silenced. When transplanted into nude mice,they gave rise to teratomas containing various tissues (FIG. 16 a).These data confirmed pluripotency of iPS cells generated by the threefactors from p53-null MEFs.

We found that iPS cells generated by the four factors including c-Mycalso showed a morphology indistinguishable from that of ES cells (FIG.14, right). However, the expressions of ES-cell markers were lower inthese cells than in ES cells (FIG. 15, right). In addition, transgeneexpression of the four factors remained active in these cells.Consistent with this observation, tumors derived from these cells innude mice largely consist of undifferentiated cells, with only smallareas of differentiated tissues (FIG. 16 b). Thus c-Myc, in the p53-nullbackground, suppresses retroviral silencing and inhibitsdifferentiation.

Example 7 Effects of p53 Suppression on Establishment of Human iPS Cells

(A) We then examined whether p53 deletion increased efficiency of humaniPS cell generation. To this end, we introduced the dominant negativemutatants of p53 (p275S or DD (Bowman T. et al. Genes Dev 10(7), 826(1996))) into adult human dermal fibroblasts (HDFs) together with thethree or four reprogramming factors. Retroviral vector for the P275Smutant was generated as described in Example 6. Retroviral vector forthe DD mutant was generated by two step PCR. The first PCR was performedwith two primer sets, p53-DD-S (cgg ata tca gcc tca aga gag cgc tgc c;SEQ ID NO:39) and p53-1223AS, and p53-DD-AS (ggc agc get ctc ttg agg ctgata tcc g; SEQ ID NO:40) and p53-1S. Retroviruses for dominant negativemutants, and the four or three reprogramming factors were produced inPLAT-E cells. For iPS cell generation, equal amounts of PLAT-Esupernatants containing each retrovirus were mixed and transduced toHDF.

We found that the numbers of iPS cell colonies markedly increased withthe two independent p53 dominant negative mutants (FIG. 17 a, b). Whentransplanted into testes of SCID mice, the human iPS cells generated bythe three factors and the p53DD mutant developed teratomas containingvarious tissues of three germ layers (FIG. 17 c).

(B) In another experiment, we examined effects of shRNA against humanp53 (shRNA2; Stewart S. A. et al. RNA 9(4), 493 (2003)). Retroviralvectors for shRNA expression, pMKO.1-puro (Addgene #8452), pMKO.1-purop53 shRNA1 (Addgene #10671) and pMKO.1-puro p53 shRNA2 (Addgene #10672;shRNA sequence is shown in SEQ ID NO:28), were obtained from Addgene.Retroviruses for shRNAs, and the four or three reprogramming factorswere produced in PLAT-E cells. For iPS cell generation, equal amounts ofPLAT-E supernatants containing each retrovirus were mixed and transducedto HDF.

We confirmed that p53 shRNA2 effectively suppressed the p53 proteinlevel (FIG. 18) in HDFs. When co-introduced with the four reprogrammingfactors, the p53 shRNA markedly increased numbers of human iPS cellcoloneis (FIG. 19). A control shRNA containing one nucleotide deletionin the antisense sequence (shRNA1) did not show such effects (FIG. 18and FIG. 19). Co-introduction of the mouse p53 suppressed the effect ofshRNA2. Similar results were obtained when co-introduced with the threereprogramming factors (FIG. 20). These data demonstrated that p53suppresses direct reprogramming not only in mice, but also in human.

Example 8 Functional Inhibition of p53 by MDM2

The effects of MDM2 that binds with and inhibits p53 were examined.Specifically, human MDM2 gene (SEQ ID NO:41) was co-introduced with thefour or three reprogramming factors into HDFs using retroviral vectors,and iPS colonies were formed in the same manner as in Example 7 (n=4).

As a result, the co-introduction of MDM2 gene improved the establishmentefficiency of iPS cells (FIG. 21). While MDM2 also interacts with andinhibits another tumor suppressor, Retinoblastoma (Rb), suppression ofRb by shRNA did not increase the efficiency of iPS cell generation.These data suggest that the effects of MDM2 are caused by suppression ofthe p53 function.

Example 9 In Vitro Differentiation Induction and Immunostaining

Next, the present inventors confirmed whether the cells generated withthe three reprogramming factors and the p53 shRNA in Example 7(B) werepluripotent by in vitro differentiation. To form embryoid bodies, thecells were harvested and transferred to poly-hydroxyethyl methacrylate(HEMA)-coated dishes and incubated for 8 days. After floating culture,the embryoid bodies formed were plated onto gelatin-coated plates andincubated for another 6 days. After incubation, the cells were fixedwith 4% paraformaldehyde and permeabilized and blocked with PBScontaining 5% normal goat serum, 1% BSA and 0.2% TritonX-100. Theexpression of differentiation markers (AFP, α-SMA, βIII-tubulin) wasexamined by immunocytochemistry. As primary antibodies,anti-α-fetoprotein (AFP) (1:100, R&D systems), anti-α-smooth muscleactin (α-SMA) (1:500, DAKO) and anti-βIII-tubulin (1:100, Chemicon) wereused. Cy3-labeled anti-mouse IgG (1:500, Chemicon) was used as secondaryantibody. Nuclei were stained with Hoechst 33342 (Invitrogen). Theresults are shown in FIG. 22. The iPS cells differentiated into threegerm layers such as endoderm (AFP), mesoderm (α-SMA) and ectoderm(PIII-tubulin). No significant difference in differentiation potentialswas found between the iPS clones.

Example 10 RT-PCR Analysis of Undifferentiated Markers andDifferentiated Markers

The expression of stem cell markers (Oct3/4, Sox2, Nanog) anddifferentiation markers (FoxA2 and Sox17 (endoderm), Msx1 (mesoderm),Map2 and Pax6 (ectoderm)) in the human iPS cells obtained in Example7(B) and the differentiated cells obtained in Example 9 were analyzed byRT-PCR using Rever Tra Ace Kit (Takara). The primer pairs used foramplifying the markers are shown in Table 1.

TABLE 1 SEQ ID Gene Sequence (5′-3′) NO: Stem cell markers Oct3/4GAC AGG GGG AGG GGA GGA GCT AGG 43 CTT CCC TCC AAC CAG TTG CCC CAA  44AC Sox2 GGG AAA TGG GAG GGG TGC AAA AGA  45 GGTTG CGT GAG TGT GGA TGG GAT TGG  46 TG NanogCAG CCC CGA TTC TTC CAC CAG TCC  47 C CGG AAG ATT CCC AGT CGG GTT CAC 48 C Differentiation markers FoxA2 TGG GAG CGG TGA AGA TGG AAG GGC  49AC TCA TGC CAG CGC CCA CGT ACG ACG  50 AC Sox17CGC TTT CAT GGT GTG GGC TAA GGA  51 CG TAG TTG GGG TGG TCC TGC ATG TGC 52 TG Msx1 CGA GAG GAC CCC GTG GAT GCA GAG 53GGC GGC CAT CTT CAG CTT CTC CAG 54 Pax6 ACC CAT TAT CCA GAT GTG TTT GCC 55 CGA G ATG GTG AAG CTG GGC ATA GGC GGC  56 AG Map2CAG GTG GCG GAC GTG TGA AAA TTG  57 AGA GTGCAC GCT GGA TCT GCC TGG GGA CTG  58 TG Internal standard Nat1ATT CTT CGT TGT CAA GCC GCC AAA   59 GTG GAGAGT TGT TTG CTG CGG AGT TGT CAT   60 CTC GTC

As a result, the cells generated with the three reprogramming factorsand the p53 shRNA expressed Nanog, endogenous Oct3/4, and endogenousSox2 at comparable levels to those in ES cells (FIG. 23, lanes U). Afterembryoid body-mediated differentiation, these cells expressed markergenes of the three germ layers (FIG. 23, lanes D). These datademonstrated that p53 suppresses direct reprogramming not only in mice,but also in human.

Example 11 Role of p21 in Improving the Efficiency of Establishment ofiPS Cells by p53 Suppression

To elucidate p53 target genes that are responsible for the observedenhancement of iPS cell generation, we compared gene expression betweenp53 wild-type MEF and p53-null MEF by DNA microarrays, and betweencontrol HDF and p53 knockdown HDF. In MEF, 1590 genes increased and 1485genes decreased >5 fold in p53-null MEF. In HDF, 290 genes increased and430 genes decreased >5 fold by p53 shRNA. Between mouse and human, eightincreased genes are common, including v-myb myeloblastosis viraloncogene homolog (MYB) and a RAS oncogenes family, RAB39B (Table 2).Twenty-seven decreased genes were common between the two species,including p53, cyclin-dependent kinase inhibitor 1A (p21, Cip1), BTGfamily, member 2 (BTG2), zinc finger, matrin type 3 (ZMAT3), and MDM2.

TABLE 2 Accession No. Gene Name Increased genes NM_000809 Homo sapiensgamma-aminobutyric acid (GABA) A receptor, alpha 4 (GABRA4) NM_182767Homo sapiens solute carrier family 6 (neutral amino acid transporter),member 15 (SLC6A15), transcript variant 1 NM_014264 Homo sapienspolo-like kinase 4 (Drosophila) (PLK4) NM_198391 Homo sapiensfibronectin leucine rich transmembrane protein 3 (FLRT3), transcriptvariant 2 NM_171998 Homo sapiens RAB39B, member RAS oncogene family(RAB39B) NM_001482 Homo sapiens glycine amidinotransferase (L-arginine:glycine amidinotransferase) (GATM), nuclear gene encoding NM_001130173Homo sapiens v-myb myeloblastosis viral oncogene homolog (avian) (MYB),transcript variant 1 NM_004004 Homo sapiens gap junction protein, beta2, 26 kDa (GJB2) Decreased genes NM_000546 Homo sapiens tumor proteinp53 (TP53), transcript variant 1 NM_000389 Homo sapiens cyclin-dependentkinase inhibitor 1A (p21, Cip1) (CDKN1A) NM_001461 Homo sapiens flavincontaining monooxygenase 5 (FMO5) NM_002474 Homo sapiens myosin, heavychain 11, smooth muscle (MYH11), transcript variant SM1A NM_006763 Homosapiens BTG family, member 2 (BTG2) NM_004455 Homo sapiens exostoses(multiple)-like 1 (EXTL1) NM_022470 Homo sapiens zinc finger, matrintype 3 (ZMAT3), transcript variant 1 NM_006536 Homo sapiens CLCA familymember 2, chloride channel regulator (CLCA2) NM_001017915 Homo sapiensinositol polyphosphate-5-phosphatase, 145 kDa (INPP5D), transcriptvariant 1 NM_006879 Homo sapiens Mdm2, transformed 3T3 cell doubleminute 2, p53 binding protein (mouse) (MDM2), transcript variantNM_004469 Homo sapiens c-fos induced growth factor (vascular endothelialgrowth factor D) (FIGF) NM_016352 Homo sapiens carboxypeptidase A4(CPA4) NM_024817 Homo sapiens thrombospondin, type I, domain containing4 (THSD4) NM_000526 Homo sapiens keratin 14 (KRT14) NM_000846 Homosapiens glutathione S-transferase A2 (GSTA2) NM_000198 Homo sapienshydroxy-delta-5-steroid dehydrogenase, 3 beta-and steroiddelta-isomerase 2 (HSD3B2) NM_032866 Homo sapiens cingulin-like 1(CGNL1) NM_000230 Homo sapiens leptin (LEP) NM_020405 Homo sapiensplexin domain containing 1 (PLXDC1) NM_004975 Homo sapiens potassiumvoltage-gated channel, Shab-related subfamily, member 1 (KCNB1)NM_001128310 Homo sapiens SPARC-like 1 (hevin) (SPARCL1), transcriptvariant 1 NM_032588 Homo sapiens tripartite motif-containing 63 (TRIM63)NM_022047 Homo sapiens differentially expressed in FDCP 6 homolog(mouse) (DEF6) NM_003322 Homo sapiens tubby like protein 1 (TULP1)NM_003012 Homo sapiens secreted frizzied-related protein 1 (SFRP1)NM_002164 Homo sapiens indoleamine-pyrrole 2,3 dioxygenase (INDO)NM_004060 Homo sapiens cyclin G1 (CCNG1), transcript variant 1

Among these we transduced four increased genes and seven decreased genesby retroviruses into HDF, together with either the four reprogrammingfactors alone, or with the four factors and the p53 shRNA. We reasonedthat if some of these target genes are responsible for the observedenhancement of iPS cell generation, forced expression of increased genesin wild-type fibroblasts would mimic the effect of p53 suppression,whereas co-expression of decreased genes and the p53 shRNA wouldcounteract the effect of p53 suppression. Among the four increasedgenes, none mimicked the effect of p53 suppression by forced expressionof MDM2, which binds to and degrades the p53 proteins (FIG. 24 a). Amongseven decreased genes, only p53, derived from mouse, and p21 effectivelycounteracted the effect of the p53 shRNA (FIG. 24 b). In addition, theforced expression of p21 markedly decreased IFS cell generation fromp53-null MEF. These data highlighted importance of p21 as a p53 targetduring IFS cell generation in both mouse and human.

Suppression of p53-p21 results in inactivation of Rb. However,suppression of Rb by shRNA did not increase the efficiency of iPS cellgeneration (FIG. 21). These data suggest that the effect of p53-p21suppression on IFS cell generation is, at least in part, attributable tomechanisms other than regulation of Rb.

The p21 protein binds to Myc and suppresses its transcriptional activity(Kitaura, H. et al., J Biol. Chem., 275(14), 10477-10483 (2000)). Thismight contribute to the increased efficiency of iPS cell generation byp53-p21 suppression. We evaluated the Myc activity in HDF by introducingluciferase reporters driven by either a p53-responsive element or a Mycresponsive element (FIG. 24 c). We confirmed the p53 activity wasreduced by the shRNA. In these knockdown cells, the Myc activity wassignificantly enhanced. The effect was stronger than that observed withthe introduction of the four factors including c-Myc. These data suggestthat the activation of Myc contributes to the enhanced iPS cellgeneration by p53-p21 suppression.

Example 12 Establishment of iPS Cells by Plasmid Introduction

Expression vector pCX-2A-Ms-OKS that expresses mouse Oct3/4, Klf4 andSox2, and expression vector pCX-Ms-cMyc that expresses mouse c-Myc wereboth prepared according to Science, 322, pp. 949-953 (2008).

Fibroblasts (MEFs) were isolated from a fetal p53 homo-deficient mousehaving the mouse Nanog reporter (13.5 days after fertilization) andwild-type mouse fetus (13.5 days after fertilization). The MEFs weresown to a 6-well culture plate (Falcon) coated with 0.1% gelatin (Sigma)at 1.3×10⁵ per well. The culture broth used was DMEM/10% FBS (DMEM(Nacalai tesque) supplemented with 10% fetal bovine serum), and thecells were cultured at 37° C. and 5% CO₂. The following day, usingFuGene6 transfection reagent (Roche) or Lipofectamin LTX (Invitrogen)and following the protocol attached to the reagent, pCX-2A-Ms-OKS andpCX-Ms-cMyc were introduced at once (day 0). Introduction was repeatedevery day up to day 7 from the introduction. On day 4 from theintroduction, the medium was changed to an LIF-supplemented ES cellculture medium (prepared by adding 15% fetal bovine serum, 2 mML-glutamine (Invitrogen), 100 μM non-essential amino acids (Invitrogen),100 μM 2-mercaptoethanol (Invitrogen), 50 U/mL penicillin (Invitrogen)and 50 μg/mL streptomycin (Invitrogen) to DMEM (Nacarai Tesque)).Subsequently, the LIF-supplemented ES cell culture medium was exchangedwith a fresh supply every two days until a colony was observable.Selection with puromycin (1.5 μg/mL) was performed from day 21 after theintroduction and GFP-positive colonies were counted on day 33 after theintroduction. Summary of the time schedule is shown in FIG. 25 a, andthe obtained number of GFP-positive colonies is shown in FIG. 25 b. When4 genes were introduced into wild-type MEF by plasmid (+/+ in FIG. 25b), no GFP-positive colony was formed. When 4 genes were introduced intop53 homo-deficient MEF by plasmid (−/− in FIG. 25 b), many GFP-positivecolonies were obtained.

To examine integration of plasmid DNAs into the genome, 16 kinds of PCRprimers capable of amplifying each part of the plasmid were designed(see Science, 322, pp. 949-953 (2008), FIG. 2A), and 11 kinds of primerstherefrom were used for Genomic PCR. The results are shown in FIG. 25 c.Amplification of exogenous DNA was not observed in 6 clones out of theobtained 12 GFP-positive clones (FIG. 25 c: upper panel). By Southernblot analysis using Oct3/4, Sox2, Klf4 and c-Myc as probes, moreover,integration of exogenous gene was not detected in those clones (FIG. 25c: lower panel). The above results reveal that these IFS cells do notintegrate pCX-2A-Ms-OKS and pCX-Ms-cMyc plasmids into the host genome.

The photographs of the obtained cells are shown in FIG. 25 d (upperleft: phase-contrast image, upper right: GFP-positive colony image,lower left: merge of phase-contrast image and GFP-positive colonyimage). Since the cells had a form morphologically indistinguishablefrom that of mouse ES cells, and were GFP-positive, establishment of iPScells was confirmed. Moreover, iPS clones free of plasmid integrationwere injected to ICR-mouse-derived blastocysts. The results are shown inFIG. 25 d, lower right panel. Judging from the hair color, adult chimeracould be produced from iPS clone. The results confirm that IFS cellsfree of plasmid integration have pluripotency.

While the present invention has been described with emphasis onpreferred embodiments, it is obvious to those skilled in the art thatthe preferred embodiments can be modified. The present invention intendsthat the present invention can be embodied by methods other than thosedescribed in detail in the present specification. Accordingly, thepresent invention encompasses all modifications encompassed in the gistand scope of the appended “CLAIMS.”

The contents disclosed in any publication cited herein, includingpatents and patent applications, are hereby incorporated in theirentireties by reference, to the extent that they have been disclosedherein.

The invention claimed is:
 1. An in vitro method of improving theefficiency of establishment of an induced pluripotent stem (iPS) cell,comprising contacting an isolated somatic cell being reprogrammed intoan iPS cell with siRNA or shRNA that inhibits p53 or DNA that encodesthe siRNA or shRNA.
 2. The method of claim 1, wherein the isolatedsomatic cell being reprogrammed into an iPS cell is contacted with siRNAthat inhibits p53 or DNA that encodes the siRNA.
 3. The method of claim1, wherein the isolated somatic cell being reprogrammed into an iPS cellis contacted with shRNA that inhibits p53 or DNA that encodes the shRNA.4. An in vitro method of producing iPS cells, comprising bringing (a)nuclear reprogramming substances or nucleic acids encoding the nuclearreprogramming substances and (b) an inhibitor of p53 function intocontact with a somatic cell, wherein the nuclear reprogrammingsubstances are (i) Oct3/4 and Klf4, (ii) Oct3/4 and c-Myc, (iii) Oct3/4,Klf4 and Sox2, (iv) Oct3/4, Klf4 and c-Myc, or (v) Oct3/4, Klf4, Sox2and c-Myc, and wherein the inhibitor of p53 function is siRNA or shRNAthat inhibits p53 or DNA that encodes the siRNA or shRNA.
 5. The methodof claim 4, wherein the nuclear reprogramming substances are Oct3/4,Klf4 and Sox2, or nucleic acids that encode the same.
 6. The method ofclaim 4, wherein the nuclear reprogramming substances are Oct3/4, Klf4,Sox2 and c-Myc, or nucleic acids that encode the same.
 7. The method ofclaim 4, wherein the somatic cell is a T cell.
 8. The method of claim 4,wherein the inhibitor of p53 function is siRNA that inhibits p53 or DNAthat encodes the siRNA.
 9. The method of claim 7, wherein the inhibitorof p53 function is shRNA that inhibits p53 or DNA that encodes theshRNA.